Adeno-associated virus serotype 1 nucleic acid sequences, vectors and host cells containing same

ABSTRACT

The nucleic acid sequences of adeno-associated virus (AAV) serotype 1 are provided, as are vectors and host cells containing these sequences and functional fragments thereof. Also provided are methods of delivering genes via AAV-1 derived vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional of U.S. patent application Ser. No.09/807,802, filed Nov. 29, 2001, which is a national phase ofPCT/US99/25694, filed Nov. 2, 1999, which claims the benefit of thepriority of U.S. Patent Application No. 60/107,114, filed Nov. 5, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This work was supported by the National Institutes of Health,grant no. P30 DK47757-06 and PO1 HD32649-04. The US government may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] This invention relates generally to viral vector, and moreparticularly, to recombinant viral vectors useful for gene delivery.Adeno-associated viruses are small, single-stranded DNA viruses whichrequire helper virus to facilitate efficient replication [K. I. Berns,Parvoviridae, the viruses and their replication, p. 1007-1041, in F. N.Fields et al., Fundamental virology, 3rd ed., vol. 2, (Lippencott-RavenPublishers, Philadelphia, Pa.) (1995)]. The 4.7 kb genome of AAV ischaracterized by two inverted terminal repeats (ITR) and two openreading frames which encode the Rep proteins and Cap proteins,respectively. The Rep reading frame encodes four proteins of molecularweight 78 kD, 68 kD, 52 kD and 40 kD. These proteins function mainly inregulating AAV replication and integration of the AAV into a host cell'schromosomes. The Cap reading frame encodes three structural proteins inmolecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD (VP3) [Berns, citedabove]. More than 80% of total proteins in AAV virion comprise VP3. Thetwo ITRs are the only cis elements essential for AAV replication,packaging and integration. There are two conformations of AAV ITRscalled “flip” and “flop”. These differences in conformation originatedfrom the replication model of adeno-associated virus which use the ITRto initiate and re-initiate the replication [R. O. Snyder et al., J.Virol., 67:6096-6104 (1993); K. I. Berns, Microbiological Reviews,54:316-329 (1990)].

[0004] AAVs have been found in many animal species, including primates,canine, fowl and human [F. A. Murphy et al., “The Classification andNomenclature of Viruses: Sixth Report of the International Committee onTaxonomy of Viruses”, Archives of Virology, (Springer-Verlag, Vienna)(1995)]. In addition to five known primate AAVs (AAV-1 to AAV-5), AAV-6,another serotype closely related to AAV-2 and AAV-1 has also beenisolated [E. A. Rutledge et al., J. Virol., 72:309-319 (1998)]. Amongall known AAV serotypes, AAV-2 is perhaps the most well-characterizedserotype, because its infectious clone was the first made [R. J.Samulski et al., Proc. Natl Acad. Sci. USA, 79:2077-2081 (1982)].Subsequently, the full sequences for AAV-3A, AAV-3B, AAV-4 and AAV-6have also been determined [Rutledge, cited above; J. A. Chiorini et al.,J. Virol., 71:6823-6833 (1997); S. Muramatsu et al., Virol., 221:208-217(1996)]. Generally, all AAVs share more than 80% homology in nucleotidesequence.

[0005] A number of unique properties make AAV a promising vector forhuman gene therapy [Muzyczka, Current Topics in Microbiology andImmunology, 158:97-129 (1992)]. Unlike other viral vectors, AAVs havenot been shown to be associated with any known human disease and aregenerally not considered pathogenic. Wild type AAV is capable ofintegrating into host chromosomes in a site specific manner [R. M. Kotinet al., Proc, Natl. Acad, Sci, USA, 87:2211-2215 (1990)—R. J. Samulski,EMBO J., 10(12):3941-3950 (1991)]. Recombinant AAV vectors can integrateinto tissue cultured cells in chromosome 19 if the rep proteins aresupplied in trans [C. Balague et al., J. Virol., 71:3299-3306 (1997); R.T. Surosky et al., J. Virol., 71:7951-7959 (1997)]. The integratedgenomes of AAV have been shown to allow long term gene expression in anumber of tissues, including, muscle, liver, and brain [K. J. Fisher,Nature Med., 3(3):306-312 (1997); R. O. Snyder et al., Nature Genetics,16:270-276 (1997); X. Xiao et al., Experimental Neurology, 144:113-124(1997); Xiao, J. Virol., 70(11):8098-8108 (1996)].

[0006] AAV-2 has been shown to be present in about 80-90% of the humanpopulation. Earlier studies showed that neutralizing antibodies forAAV-2 are prevalent [W. P. Parks et al., J. Virol., 2:716-722 (1970)].The presence of such antibodies may significantly decrease theusefulness of AAV vectors based on AAV-2 despite its other merits. Whatare needed in the art are vectors characterized by the advantages ofAAV-2, including those described above, without the disadvantages,including the presence of neutralizing antibodies.

SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides an isolated AAV-1 nucleicacid molecule which is selected from among SEQ ID NO: 1, the strandcomplementary to SEQ ID NO: 1, and cDNA and RNA sequences complementaryto SEQ ID NO: 1 and its complementary strand.

[0008] In another aspect, the present invention provides AAV ITRsequences, which include the 5′ ITR sequences, nt 1 to 143 of SEQ ID NO:1; the 3′ ITR sequences, nt 4576 to 4718 of SEQ ID NO: 1, and fragmentsthereof.

[0009] In yet another aspect, the present invention provides arecombinant vector comprising an AAV-1 ITR and a selected transgene.Preferably, the vector comprises both the 5′ and 3′ AAV-1 ITRs betweenwhich the selected transgene is located.

[0010] In still another aspect, the invention provides a recombinantvector comprising an AAV-1 P5 promoter having the sequence of nt 236 to299 of SEQ ID NO: 1 or a functional fragment thereof.

[0011] In a further aspect, the present invention provides a nucleicacid molecule encoding an AAV-1 rep coding region and an AAV-1 capcoding region. In still another aspect, the present invention provides ahost cell transduced with a recombinant viral vector of the invention.The invention further provides a host cell stably transduced with anAAV-1 P5 promoter of the invention.

[0012] In still a further aspect, the present invention provides apharmaceutical composition comprising a carrier and a vector of theinvention.

[0013] In yet another aspect, the present invention provides a methodfor AAV-mediated delivery of a transgene to a host involving the step ofdelivering to a selected host a recombinant viral vector comprising aselected transgene under the control of sequences which directexpression thereof and an adeno-associated virus 1 (AAV-1) virion.

[0014] In another aspect, the invention provides a method for in vitroproduction of a selected gene product using a vector of the invention.

[0015] Other aspects and advantages of the invention will be readilyapparent to one of skill in the art from the detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1C illustrate the alignment of nucleotides of AAV-1 [SEQID NO: 1], AAV-2 [SEQ ID NO: 18] and AAV-6 [SEQ ID NO: 19]. Thealignment was done with MacVector 6.0. The full sequences of AAV-1 areshown in the top line. Nucleotides in AAV-2 and AAV-6 identical to AAV-1are symbolized by “.” and gaps by “-”. Some of the conserved featuresamong AAVs are marked in this figure. Note the 3′ ITRs of AAV-1 andAAV-6 are shown in different orientations.

[0017]FIG. 2 illustrates the predicted secondary structure of AAV-1 ITR.The nucleotides in AAV-2 and AAV-6 are shown in italic and boldrespectively.

[0018]FIG. 3A illustrates a hypothesis of how AAV-6 arose from thehomologous recombination between AAV-1 and AAV-2. The major elements ofAAV-1 are indicated in the graph. A region that is shared between AAV-1,AAV-2 and AAV-6 is shown in box with waved lines.

[0019]FIG. 3B is a detailed illustration of a 71 bp homologous regionamong AAV-1, AAV-2 and AAV-6. Nucleotides that differ among theseserotypes are indicated by arrows.

[0020]FIG. 4A is a bar chart illustrating expression levels of humanalpha 1 anti-trypsin (α1AT) in serum following delivery of hAAT viarecombinant AAV-1 and recombinant AAV-2 viruses.

[0021]FIG. 4B is a bar chart illustrating expression levels oferythropoietin (epo) in serum following delivery of the epo gene viarecombinant AAV-1 and recombinant AAV-2 viruses.

[0022]FIG. 5A is a bar chart illustrating expression levels of α1AT inliver following delivery of α1AT as described in Example 7.

[0023]FIG. 5B is a bar chart demonstrating expression levels of epo inliver following delivery of epo as described in Example 7.

[0024]FIG. 5C is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-1 following delivery of α1AT or epo to liver asdescribed in Example 7. FIG. 5D is a bar chart demonstratingneutralizing antibodies (NAB) directed to AAV-2 following delivery ofα1AT or epo to liver as described in Example 7.

[0025]FIG. 6A is a bar chart illustrating expression levels of α1AT inmuscle following delivery of α1AT as described in Example 7.

[0026]FIG. 6B is a bar chart demonstrating expression levels of epo inmuscle following delivery of epo as described in Example 7.

[0027]FIG. 6C is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-1 following delivery of α1AT or epo to muscle asdescribed in Example 7.

[0028]FIG. 6D is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-2 following delivery of α1AT or epo to muscle asdescribed in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides novel nucleic acid sequences foran adeno-associated virus of serotype 1 (AAV-1). Also provided arefragments of these AAV-1 sequences. Among particularly desirable AAV-1fragments are the inverted terminal repeat sequences (ITRs), rep andcap. Each of these fragments may be readily utilized, e.g., as acassette, in a variety of vector systems and host cells. Such fragmentsmay be used alone, in combination with other AAV-1 sequences orfragments, or in combination with elements from other AAV or non-AAVviral sequences. In one particularly desirable embodiment, a cassettemay contain the AAV-1 ITRs of the invention flanking a selectedtransgene. In another desirable embodiment, a cassette may contain theAAV-1 rep and/or cap proteins, e.g., for use in producing recombinant(rAAV) virus.

[0030] Thus, the AAV-1 sequences and fragments thereof are useful inproduction of rAAV, and are also useful as antisense delivery vectors,gene therapy vectors, or vaccine vectors. The invention further providesnucleic acid molecules, gene delivery vectors, and host cells whichcontain the AAV-1 sequences of the invention. Also provided a novelmethods of gene delivery using AAV vectors.

[0031] As described herein, the vectors of the invention containing theAAV-1 capsid proteins of the invention are particularly well suited foruse in applications in which the neutralizing antibodies diminish theeffectiveness of other AAV serotype based vectors, as well as otherviral vectors. The rAAV vectors of the invention are particularlyadvantageous in rAAV readministration and repeat gene therapy.

[0032] These and other embodiments and advantages of the invention aredescribed in more detail below. As used throughout this specificationand the claims, the term “comprising” is inclusive of other components,elements, integers, steps and the like.

[0033] I. AAV1 Nucleic Acid and Protein Sequences

[0034] The AAV-1 nucleic acid sequences of the invention include the DNAsequences of SEQ ID NO: 1 (FIGS. 1A-1C), which consists of 4718nucleotides. The AAV-1 nucleic acid sequences of the invention furtherencompass the strand which is complementary to SEQ ID NO: 1, as well asthe RNA and cDNA sequences corresponding to SEQ ID NO: 1 and itscomplementary strand. Also included in the nucleic acid sequences of theinvention are natural variants and engineered modifications of SEQ IDNO: 1 and its complementary strand. Such modifications include, forexample, labels which are known in the art, methylation, andsubstitution of one or more of the naturally occurring nucleotides withan analog.

[0035] Further included in this invention are nucleic acid sequenceswhich are greater than 85%, preferably at least about 90%, morepreferably at least about 95%, and most preferably at least about 98-99%identical or homologous to SEQ ID NO:1.

[0036] The term “percent sequence identity” or “identical” in thecontext of nucleic acid sequences refers to the residues in the twosequences which are the same when aligned for maximum correspondence.The length of sequence identity comparison may be over the full-lengthsequence, or a fragment at least about nine nucleotides, usually atleast about 20-24 nucleotides, at least about 28-32 nucleotides, andpreferably at least about 36 or more nucleotides. There are a number ofdifferent algorithms known in the art which can be used to measurenucleotide sequence identity. For instance, polynucleotide sequences canbe compared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, 1990, hereinincorporated by reference). For instance, percent sequence identitybetween nucleic acid sequences can be determined using Fasta with itsdefault parameters (a word size of 6 and the NOPAM factor for thescoring matrix) as provided in GCG Version 6.1, herein incorporated byreference.

[0037] The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95-99% of the sequence.

[0038] Also included within the invention are fragments of SEQ ID NO: 1,its complementary strand, cDNA and RNA complementary thereto. Suitablefragments are at least 15 nucleotides in length, and encompassfunctional fragments which are of biological interest. Certain of thesefragments may be identified by reference to FIGS. 1A-1C. Examples ofparticularly desirable functional fragments include the AAV-1 invertedterminal repeat (ITR) sequences of the invention. In contrast to the 145nt ITRs of AAV-2, AAV-3, and AAV-4, the AAV-1 ITRs have been found toconsist of only 143 nucleotides, yet advantageously are characterized bythe T-shaped hairpin structure which is believed to be responsible forthe ability of the AAV-2 ITRs to direct site-specific integration. Inaddition, AAV-1 is unique among other AAV serotypes, in that the 5′ and3′ ITRs are identical. The full-length 5′ ITR sequences of AAV-1 areprovided at nucleotides 1-143 of SEQ ID NO: 1 (FIG. 1A) and thefull-length 3′ ITR sequences of AAV-1 are provided at nt 4576-4718 ofSEQ ID NO: 1 (FIG. 1C). One of skill in the art can readily utilize lessthan the full-length 5′ and/or 3′ ITR sequences for various purposes andmay construct modified ITRs using conventional techniques, e.g., asdescribed for AAV-2 ITRs in Samulski et al, Cell, 33:135-143 (1983).

[0039] Another desirable functional fragment of the AAV-1 genome is theP5 promoter of AAV-1 which has sequences unique among AAV P5 promoters,while maintaining critical regulatory elements and functions. Thispromoter is located within nt 236-299 of SEQ ID NO: 1 (FIG. 1A). Otherexamples of functional fragments of interest include the sequences atthe junction of the rep/cap, e.g., the sequences spanning nt 2306-2223,as well as larger fragments which encompass this junction which maycomprise 50 nucleotides on either side of this junction. Still otherexamples of functional fragments include the sequences encoding the repproteins. Rep 78 is located in the region of nt 334-2306 of SEQ ID NO:1; Rep 68 is located in the region of nt 334-2272, and contains anintron spanning nt 1924-2220 of SEQ ID NO: 1. Rep 52 is located in theregion of nt 1007-2304 of SEQ ID NO: 1; rep 40 is located in the regionof nt 1007-2272, and contains an intron spanning nt 1924-2246 of SEQ IDNO: 1. Also of interest are the sequences encoding the capsid proteins,VP 1 [nt 2223-4431 of SEQ ID NO: 1], VP2 [nt 2634-4432 of SEQ ID NO: 1]and VP3 [nt 2829-4432 of SEQ ID NO: 1]. Other fragments of interest mayinclude the AAV-1 P 19 sequences, AAV-1 P40 sequences, the rep bindingsite, and the terminal resolute site (TRS).

[0040] The invention further provides the proteins and fragments thereofwhich are encoded by the AAV-1 nucleic acids of the invention.Particularly desirable proteins include the rep and cap proteins, whichare encoded by the nucleotide sequences identified above. These proteinsinclude rep 78 [SEQ ID NO:5], rep 68 [SEQ ID NO:7], rep 52 [SEQ IDNO:9], rep 40 [SEQ ID NO: 11], vp1 [SEQ ID NO: 13], vp2 [SEQ ID NO: 15],and vp3 [SEQ ID NO: 17] and functional fragments thereof while thesequences of the rep and cap proteins have been found to be closelyrelated to those of AAV-6, there are differences in the amino acidsequences (see Table 1 below), as well as differences in the recognitionof these proteins by the immune system. However, one of skill in the artmay readily select other suitable proteins or protein fragments ofbiological interest. Suitably, such fragments are at least 8 amino acidsin length. However, fragments of other desired lengths may be readilyutilized. Such fragments may be produced recombinantly or by othersuitable means, e.g., chemical synthesis.

[0041] The sequences, proteins, and fragments of the invention may beproduced by any suitable means, including recombinant production,chemical synthesis, or other synthetic means. Such production methodsare within the knowledge of those of skill in the art and are not alimitation of the present invention.

[0042] II. Viral Vectors

[0043] In another aspect, the present invention provides vectors whichutilize the AAV-1 sequences of the invention, including fragmentsthereof, for delivery of a heterologous gene or other nucleic acidsequences to a target cell. Suitably, these heterologous sequences(i.e., a transgene) encode a protein or gene product which is capable ofbeing expressed in the target cell. Such a transgene may be constructedin the form of a “minigene”. Such a “minigene” includes selectedheterologous gene sequences and the other regulatory elements necessaryto transcribe the gene and express the gene product in a host cell.Thus, the gene sequences are operatively linked to regulatory componentsin a manner which permit their transcription. Such components includeconventional regulatory elements necessary to drive expression of thetransgene in a cell containing the viral vector. The minigene may alsocontain a selected promoter which is linked to the transgene andlocated, with other regulatory elements, within the selected viralsequences of the recombinant vector.

[0044] Selection of the promoter is a routine matter and is not alimitation of this invention. Useful promoters may be constitutivepromoters or regulated (inducible) promoters, which will enable controlof the timing and amount of the transgene to be expressed. For example,desirable promoters include the cytomegalovirus (CMV) immediate earlypromoter/enhancer [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the Rous sarcoma virus LTR promoter/enhancer, and the chickencytoplasmic β-actin promoter [T. A. Kost et al, Nucl, Acids Res.,11(23):8287 (1983)]. Still other desirable promoters are the albuminpromoter and an AAV P5 promoter. Optionally, the selected promoter isused in conjunction with a heterologous enhancer, e.g., the β-actinpromoter may be used in conjunction with the CMV enhancer. Yet othersuitable or desirable promoters and enhancers may be selected by one ofskill in the art.

[0045] The minigene may also desirably contain nucleic acid sequencesheterologous to the viral vector sequences including sequences providingsignals required for efficient polyadenylation of the transcript (poly-Aor pA) and introns with functional splice donor and acceptor sites. Acommon poly-A sequence which is employed in the exemplary vectors ofthis invention is that derived from the papovavirus SV-40. The poly-Asequence generally is inserted in the minigene downstream of thetransgene sequences and upstream of the viral vector sequences. A commonintron sequence is also derived from SV-40, and is referred to as theSV40 T intron sequence. A minigene of the present invention may alsocontain such an intron, desirably located between the promoter/enhancersequence and the transgene. Selection of these and other common vectorelements are conventional [see, e.g., Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d edit., Cold Spring Harbor Laboratory,New York (1989) and references cited therein] and many such sequencesare available from commercial and industrial sources as well as fromGenebank.

[0046] The selection of the transgene is not a limitation of the presentinvention. Suitable transgenes may be readily selected from amongdesirable reporter genes, therapeutic genes, and optionally, genesencoding immunogenic polypeptides. Examples of suitable reporter genesinclude β-galactosidase (β-gal), an alkaline phosphatase gene, and greenfluorescent protein (GFP). Examples of therapeutic genes include,cytokines, growth factors, hormones, and differentiation factors, amongothers. The transgene may be readily selected by one of skill in theart. See, e.g., WO 98/09657, which identifies other suitable transgenes.

[0047] Suitably, the vectors of the invention contain, at a minimum,cassettes which consist of fragments of the AAV-1 sequences andproteins. In one embodiment, a vector of the invention comprises aselected transgene, which is flanked by a 5′ ITR and a 3′ ITR, at leastone of which is an AAV-1 ITR of the invention. Suitably, vectors of theinvention may contain a AAV-1 P5 promoter of the invention. In yetanother embodiment, a plasmid or vector of the invention contains AAV-1rep sequences. In still another embodiment, a plasmid or vector of theinvention contains at least one of the AAV-1 cap proteins of theinvention. Most suitably, these AAV-1-derived vectors are assembled intoviral vectors, as described herein.

[0048] A. AAV Viral Vectors

[0049] In one aspect, the present invention provides a recombinant AAV-1viral vector produced using the AAV-1 capsid proteins of the invention.The packaged rAAV-1 virions of the invention may contain, in addition toa selected minigene, other AAV-1 sequences, or may contain sequencesfrom other AAV serotypes.

[0050] Methods of generating rAAV virions are well known and theselection of a suitable method is not a limitation on the presentinvention. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) andU.S. Pat. No. 5,478,745. In one suitable method, a selected host cell isprovided with the AAV sequence encoding a rep protein, the gene encodingthe AAV cap protein and with the sequences for packaging and subsequentdelivery. Desirably, the method utilizes the sequences encoding theAAV-1 rep and/or cap proteins of the invention.

[0051] In one embodiment, the rep/cap genes and the sequences fordelivery are supplied by co-transfection of vectors carrying these genesand sequences. In one currently preferred embodiment, a cis (vector)plasmid, a trans plasmid containing the rep and cap genes, and a plasmidcontaining the adenovirus helper genes are co-transfected into asuitable cell line, e.g., 293. Alternatively, one or more of thesefunctions may be provided in trans via separate vectors, or may be foundin a suitably engineered packaging cell line.

[0052] An exemplary cis plasmid will contain, in 5′ to 3′ order, AAV 5′ITR, the selected transgene, and AAV 3′ ITR. In one desirableembodiment, at least one of the AAV ITRs is a 143 nt AAV-1 ITR. However,other AAV serotype ITRs may be readily selected. Suitably, thefull-length ITRs are utilized. However, one of skill in the art canreadily prepare modified AAV ITRs using conventional techniques.Similarly, methods for construction of such plasmids is well known tothose of skill in the art.

[0053] A trans plasmid for use in the production of the rAAV-1 virionparticle may be prepared according to known techniques. In one desiredembodiment, this plasmid contains the rep and cap proteins of AAV-1, orfunctional fragments thereof. Alternatively, the rep sequences may befrom another selected AAV serotype.

[0054] The cis and trans plasmid may then be co-transfected with awild-type helper virus (e.g., Ad2, Ad5, or a herpesvirus), or moredesirably, a replication—defective adenovirus, into a selected hostcell. Alternatively, the cis and trans plasmid may be co-transfectedinto a selected host cell together with a transfected plasmid whichprovides the necessary helper functions. Selection of a suitable hostcell is well within the skill of those in the art and include suchmammalian cells as 293 cells, HeLa cells, among others.

[0055] Alternatively, the cis plasmid and, optionally the trans plasmid,may be transfected into a packaging cell line which provides theremaining helper functions necessary for production of a rAAV containingthe desired AAV-1 sequences of the invention. An example of a suitablepackaging cell line, where an AAV-2 capsid is desired, is B-50, whichstably expresses AAV-2 rep and cap genes under the control of ahomologous P5 promoter. This cell line is characterized by integrationinto the cellular chromosome of multiple copies (at least 5 copies) ofP5-rep-cap gene cassettes in a concatomer form. This B-50 cell line wasdeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Sep. 18, 1997 under AccessionNo. CRL-12401 pursuant to the provisions of the Budapest Treaty.However, the present invention is not limited as to the selection of thepackaging cell line.

[0056] Exemplary transducing vectors based on AAV-1 capsid proteins havebeen tested both in vivo and in vitro, as described in more detail inExample 4. In these studies, it was demonstrated that recombinant AAVvector with an AAV-1 virion can transduce both mouse liver and muscle.These, and other AAV-1 based gene therapy vectors which may be generatedby one of skill in the art are beneficial for gene delivery to selectedhost cells and gene therapy patients since the neutralization antibodiesof AAV-1 present in much of the human population exhibit differentpatterns from other AAV serotypes and therefore do not neutralize theAAV-1 virions. One of skill in the art may readily prepare other rAAVviral vectors containing the AAV-1 capsid proteins provided herein usinga variety of techniques known to those of skill in the art. One maysimilarly prepare still other rAAV viral vectors containing AAV-1sequence and AAV capsids of another serotype.

[0057] B. Other Viral Vectors

[0058] One of skill in the art will readily understand that the AAV-1sequences of the invention can be readily adapted for use in these andother viral vector systems for in vitro, ex vivo or in vivo genedelivery. Particularly well suited for use in such viral vector systemsare the AAV-1 ITR sequences, the AAV-1 rep, the AAV-1 cap, and the AAV-1P5 promoter sequences.

[0059] For example, in one desirable embodiment, the AAV-1 ITR sequencesof the invention may be used in an expression cassette which includesAAV-1 5′ ITR, a non-AAV DNA sequences of interest (e.g., a minigene),and 3′ ITR and which lacks functional rep/cap. Such a cassettecontaining an AAV-1 ITR may be located on a plasmid for subsequenttransfection into a desired host cell, such as the cis plasmid describedabove. This expression cassette may further be provided with an AAVcapsid of a selected serotype to permit infection of a cell or stablytransfected into a desired host cell for packaging of rAAV virions. Suchan expression cassette may be readily adapted for use in other viralsystems, including adenovirus systems and lentivirus systems. Methods ofproducing Ad/AAV vectors are well known to those of skill in the art.One desirable method is described in PCT/US95/14018. However, thepresent invention is not limited to any particular method.

[0060] Another aspect of the present invention is the novel AAV-1 P5promoter sequences which are located in the region spanning nt 236-299of SEQ ID NO: 1. This promoter is useful in a variety of viral vectorsfor driving expression of a desired transgene.

[0061] Similarly, one of skill in the art can readily select otherfragments of the AAV-1 genome of the invention for use in a variety ofvector systems. Such vectors systems may include, e.g., lentiviruses,retroviruses, poxviruses, vaccinia viruses, and adenoviral systems,among others. Selection of these vector systems is not a limitation ofthe present invention.

[0062] C. Host Cells And Packaging Cell Lines

[0063] In yet another aspect, the present invention provides host cellswhich may be transiently transfected with AAV-1 nucleic acid sequencesof the invention to permit expression of a desired transgene orproduction of a rAAV particle. For example, a selected host cell may betransfected with the AAV-1 P5 promoter sequences and/or the AAV-1 5′ ITRsequences using conventional techniques. Providing AAV helper functionsto the transfected cell lines of the invention results in packaging ofthe rAAV as infectious rAAV particles. Such cell lines may be producedin accordance with known techniques [see, e.g., U.S. Pat. No.5,658,785], making use of the AAV-1 sequences of the invention.

[0064] Alternatively, host cells of the invention may be stablytransfected with a rAAV expression cassette of the invention, and withcopies of AAV-1 rep and cap genes. Suitable parental cell lines includemammalian cell lines and it may be desirable to select host cells fromamong non-simian mammalian cells. Examples of suitable parental celllines include, without limitation, HeLa [ATCC CCL 2], A549 [ATCCAccession No. CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72]and W138 [CCL 75] cells. These cell lines are all available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 USA. Other suitable parent cell lines may be obtainedfrom other sources and may be used to construct stable cell linescontaining the P5 and/or AAV rep and cap sequences of the invention.

[0065] Recombinant vectors generated as described above are useful fordelivery of the DNA of interest to cells.

[0066] III. Methods Of Delivering Genes Via AAV-1 Derived Vectors

[0067] In another aspect, the present invention provides a method fordelivery of a transgene to a host which involves transfecting orinfecting a selected host cell with a recombinant viral vector generatedwith the AAV-1 sequences (or functional fragments thereof) of theinvention. Methods for delivery are well known to those of skill in theart and are not a limitation of the present invention.

[0068] In one desirable embodiment, the invention provides a method forAAV-mediated delivery of a transgene to a host. This method involvestransfecting or infecting a selected host cell with a recombinant viralvector containing a selected transgene under the control of sequenceswhich direct expression thereof and AAV-1 capsid proteins.

[0069] Optionally, a sample from the host may be first assayed for thepresence of antibodies to a selected AAV serotype. A variety of assayformats for detecting neutralizing antibodies are well known to those ofskill in the art. The selection of such an assay is not a limitation ofthe present invention. See, e.g., Fisher et al, Nature Med.,3(3):306-312 (March 1997) and W. C. Manning et al, Human Gene Therapy,9:477-485 (Mar. 1, 1998). The results of this assay may be used todetermine which AAV vector containing capsid proteins of a particularserotype are preferred for delivery, e.g., by the absence ofneutralizing antibodies specific for that capsid serotype.

[0070] In one aspect of this method, the delivery of vector with AAV-1capsid proteins may precede or follow delivery of a gene via a vectorwith a different serotype AAV capsid protein. Thus, gene delivery viarAAV vectors may be used for repeat gene delivery to a selected hostcell. Desirably, subsequently administered rAAV vectors carry the sametransgene as the first rAAV vector, but the subsequently administeredvectors contain capsid proteins of serotypes which differ from the firstvector. For example, if a first vector has AAV-2 capsid proteins,subsequently administered vectors may have capsid proteins selected fromamong the other serotypes, including AAV-1, AAV-3A, AAV-3B, AAV-4 andAAV-6.

[0071] Thus, a rAAV-1-derived recombinant viral vector of the inventionprovides an efficient gene transfer vehicle which can deliver a selectedtransgene to a selected host cell in vivo or ex vivo even where theorganism has neutralizing antibodies to one or more AAV serotypes. Thesecompositions are particularly well suited to gene delivery fortherapeutic purposes. However, the compositions of the invention mayalso be useful in immunization. Further, the compositions of theinvention may also be used for production of a desired gene product invitro.

[0072] The above-described recombinant vectors may be delivered to hostcells according to published methods. An AAV viral vector bearing theselected transgene may be administered to a patient, preferablysuspended in a biologically compatible solution or pharmaceuticallyacceptable delivery vehicle. A suitable vehicle includes sterile saline.Other aqueous and non-aqueous isotonic sterile injection solutions andaqueous and non-aqueous sterile suspensions known to be pharmaceuticallyacceptable carriers and well known to those of skill in the art may beemployed for this purpose.

[0073] The viral vectors are administered in sufficient amounts totransfect the cells and to provide sufficient levels of gene transferand expression to provide a therapeutic benefit without undue adverseeffects, or with medically acceptable physiological effects, which canbe determined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the liver, oral, intranasal,intravenous, intramuscular, subcutaneous, intradermal, and otherparental routes of administration. Routes of administration may becombined, if desired.

[0074] Dosages of the viral vector will depend primarily on factors suchas the condition being treated, the age, weight and health of thepatient, and may thus vary among patients. For example, atherapeutically effective human dosage of the viral vector is generallyin the range of from about 1 ml to about 100 ml of solution containingconcentrations of from about 1×10⁹ to 1×10¹⁶ genomes virus vector. Apreferred human dosage may be about 1×10¹³ to 1×10¹⁶ AAV genomes. Thedosage will be adjusted to balance the therapeutic benefit against anyside effects and such dosages may vary depending upon the therapeuticapplication for which the recombinant vector is employed. The levels ofexpression of the transgene can be monitored to determine the frequencyof dosage resulting in viral vectors, preferably AAV vectors containingthe minigene. Optionally, dosage regimens similar to those described fortherapeutic purposes may be utilized for immunization using thecompositions of the invention. For in vitro production, a desiredprotein may be obtained from a desired culture following transfection ofhost cells with a rAAV containing the gene encoding the desired proteinand culturing the cell culture under conditions which permitsexpression. The expressed protein may then be purified and isolated, asdesired. Suitable techniques for transfection, cell culturing,purification, and isolation are known to those of skill in the art.

[0075] The following examples illustrate several aspects and embodimentsof the invention.

EXAMPLE 1 Generation of Infectious Clone of AAV-1

[0076] The replicated form DNA of AAV-1 was extracted from 293 cellsthat were infected by AAV-1 and wild type adenovirus type 5.

[0077] A. Cell Culture and Virus

[0078] AAV-free 293 cells and 84-31 cells were provided by the humanapplication laboratory of the University of Pennsylvania. These cellswere cultured in Dulbecco's Modified Eagle Medium with 10% fetal bovineserum (Hyclone), penicillin (100 U/ml) and streptomycin at 37° C. in amoisturized environment supplied with 5% CO₂. The 84-31 cell lineconstitutively expresses adenovirus genes E1a, E1b, E4/ORF6, and hasbeen described previously [K. J. Fisher, J. Virol., 70:520-532 (1996)].AAV-1 (ATCC VR-645) seed stock was purchased from American Type CultureCollection (ATCC, Manassas, Va.). AAV viruses were propagated in 293cells with wild type Ad5 as a helper virus.

[0079] B. Recombinant AAV Generation

[0080] The recombinant AAV viruses were generated by transfection usingan adenovirus free method. Briefly, the cis plasmid (with AAV ITR),trans plasmid (with AAV rep gene and cap gene) and helper plasmid(pFΔ13, with essential regions from the adenovirus genome) weresimultaneously co-transfected into 293 cells in a ratio of 1:1:2 bycalcium phosphate precipitation. The pFΔ13 helper plasmid has an 8 kbdeletion in the adenovirus E2B region and has deletions in most of thelate genes. This helper plasmid was generated by deleting the RsrIIfragment from pFG140 (Microbix, Canada). Typically, 50 μg of DNA(cis:trans:PFA13 at ratios of 1:1:2, respectively) was transfected ontoa 15 cm tissue culture dish. The cells were harvested 96 hourspost-transfection, sonicated and treated with 0.5% sodium deoxycholate(37° C. for 10 min). Cell lysates were then subjected to two rounds of aCsCl gradient. Peak fractions containing AAV vector were collected,pooled, and dialyzed against PBS before injecting into animals. To makerAAV virus with AAV-1 virion, the pAVIH or p5E18 (2/1) was used as thetrans plasmid to provide rep and cap function.

[0081] For the generation of rAAV based on AAV-2, p5E18 was used as thetrans plasmid since it greatly improved the rAAV yield. This plasmid,p5E18(2/2), expresses AAV-2 Rep and Cap and contains a P5 promoterrelocated to a position 3′ to the Cap gene, thereby minimizingexpression of Rep78 and Rep68. The strategy was initially described byLi et al, J. Virol., 71:5236-5243 (1997). P5E18(2/2) was constructed inthe following way. The previously described pMMTV-trans vector (i.e.,the mouse mammary tumor virus promoter substituted for the P5 promoterin an AAV-2-based vector) was digested with SmaI and ClaI, filled inwith the Klenow enzyme, and then recircularized with DNA ligase. Theresulting construct was digested with XbaI, filled in, and ligated tothe blunt-ended BamHI-XbaI fragment from pCR-p5, constructed in thefollowing way. The P5 promoter of AAV was amplified by PCR and theamplified fragment was subsequently cloned into pCR2.1 (Invitrogen) toyield pCR-P5. The helper plasmid pAV1H was constructed by cloning theBfaI fragment of pAAV-2 into pBluescript II-SK(+) at the BcorV and SmaIsites. The 3.0-kbXbaI-KpnI fragment from p5E18(2/2), the 2.3-kbXbaI-KpnIfragment from pAV1H, and the 1.7-kb KpnI fragment from p5E18(2/2) wereincorporated into a separate plasmid P5E18(2/1), which contains AAV-2Rep, AAV-1 Cap, and the AAV-2 P5 promoter located 3′ to the Cap gene.Plasmid p5E18(2/1) produced 10- to 20-fold higher quantities of thevector than pAV1H (i.e., 10¹² genomes/50 15-cm² plates).

[0082] C. DNA Techniques

[0083] Hirt DNA extraction was performed as described in the art withminor modification [R. J. Samulski et al., Cell, 33:135-143 (1983)].More particularly, Hirst solution without SDS was used instead of usingoriginal Hirt solution containing SDS. The amount of SDS present in theoriginal Hirst solution was added after the cells had been fullysuspended. To construct AAV-1 infectious clone, the Hirt DNA from AAV-1infected 293 cells was repaired with Klenow enzyme (New England Biolabs)to ensure the ends were blunt. The treated AAV-1 Hirt DNA was thendigested with BamHI and cloned into three vectors, respectively. Theinternal BamHI was cloned into pBlueScript II-SK+ cut with BamHI to getpAV1-BM. The left and right fragments were cloned into pBlueScriptII-SK+ cut with BamHI+EcoRV to obtain pAV1-BL and pAV1-BR, respectively.The AAV sequence in these three plasmids were subsequently assembledinto the same vector to get AAV-1 infectious clone pAAV-1. The helperplasmid for recombinant AAV-1 virus generation was constructed bycloning the Bfa I fragment of pAAV-1 into pBlueScript II-SK+ at theEcoRV site.

[0084] Analysis of the Hirt DNA revealed three bands, a dimer at 9.4 kb,a monomer at 4.7 kb and single-stranded DNA at 1.7 kb, which correlatedto different replication forms of AAV-1. The monomer band was excisedfrom the gel and then digested with BamHI. This resulted in threefragments of 1.1 kb, 0.8 kb and 2.8 kb. This pattern is in accordancewith the description by Bantel-schaal and zur Hausen, Virol.,134(1):52-63 (1984). The 1.1 kb and 2.8 kb BamHI fragments were clonedinto pBlueScript-KS(+) at BamHI and EcoRV site. The internal 0.8 kbfragment was cloned into BamHI site of pBlueScript-KS(+).

[0085] These three fragments were then subcloned into the same constructto obtain a plasmid (pAAV-1) that contained the full sequence of AAV-1.The pAAV-1 was then tested for its ability to rescue from the plasmidbackbone and package infectious virus. The pAAV-1 was then transfectedto 293 cells and supplied with adenovirus type as helper at MOI 10. Thevirus supernatant was used to reinfect 293 cells.

[0086] For Southern blot analysis, Hirt DNA was digested with DpnI toremove bacteria-borne plasmid and probed with internal BamHI fragment ofAAV-1. The membrane was then washed at high stringency conditions, whichincluded: twice 30 minutes with 2×SSC, 0.1% SDS at 65° C. and twice 30minutes with 0.1×SSC, 0.1% SDS at 65° C. The membrane was then analyzedby both phosphor image and X-ray autoradiography. The results confirmedthat pAAV-1 is indeed an infectious clone of AAV serotype 1.

EXAMPLE 2 Sequencing Analysis of AAV-1

[0087] The entire AAV-1 genome was then determined by automaticsequencing and was found to be 4718 nucleotides in length (FIGS. 1A-1C).For sequencing, an ABI 373 automatic sequencer as used to determine thesequences for all plasmids and PCR fragments related to this study usingthe FS dye chemistry. All sequences were confirmed by sequencing bothplus and minus strands. These sequences were also confirmed bysequencing two independent clones of pAV-BM, pAV-BL and pAV-BR. Sincethe replicated form of AAV-1 DNA served as the template for sequencedetermination, these sequences were also confirmed by sequencing aseries of PCR products using original AAV-1 seed stock as a template.

[0088] The length of AAV-1 was found to be within the range of the otherserotypes: AAV-3 (4726 nucleotides), AAV-4 (4774 nucleotides), AAV-2(4681 nucleotides), and AAV-6 (4683 nucleotides).

[0089] The AAV-1 genome exhibited similarities to other serotypes ofadeno-associated viruses. Overall, it shares more than 80% identity withother known AAV viruses as determined by the computer program Megalignusing default settings [DNASTAR, Madison, Wis.]. The key features inAAV-2 can also be found in AAV-1. First, AAV-1 has the same type ofinverted terminal repeat which is capable of forming T-shaped hairpinstructures, despite the differences at the nucleotide level (FIGS. 2 and3). The sequences of right ITRs and left ITRs of AAV-1 are identical.The AAV TR sequence is subdivided into A, A′, B, B′, C, C′, D and D′[Bern, cited above].

[0090] These AAV ITR sequences are also virtually the same as thosefound in AAV-6 right ITR, there being one nucleotide difference in eachof A and A′ sequence, and the last nucleotide of the D sequence. Second,the AAV-2 rep binding motif [GCTCGCTCGCTCGCTG (SEQ ID NO: 20)] is wellconserved. Such motif can also be found in the human chromosome 19 AAV-2pre-integration region. Finally, non-structural and structural codingregions, and regulatory elements similar to those of other AAV serotypesalso exist in AAV-1 genome.

[0091] Although the overall features of AAV terminal repeats are verymuch conserved, the total length of the AAV terminal repeat exhibitsdivergence. The terminal repeat of AAV-1 consists of 143 nucleotideswhile those of AAV-2, AAV-3, and AAV-4 are about 145 or 146 nucleotides.The loop region of AAV-1 ITR most closely resembles that of AAV-4 inthat it also uses TCT instead of the TTT found in AAV-2 and AAV-3. Thepossibility of sequencing error was eliminated using restriction enzymedigestion, since these three nucleotides are part of the SacI site(gagctc; nt 69-74 of SEQ ID NO: 1). The p5 promoter region of AAV-1shows more variations in nucleotide sequences with other AAV serotypes.However, it still maintains the critical regulatory elements. The twocopies of YY1 [See, FIGS. 1A-1C] sites seemed to be preserved in allknown AAV serotypes, which have been shown to be involved in regulatingAAV gene expression. In AAV-4, there are 56 additional nucleotidesinserted between YY1 and E-box/USF site, while in AAV-1, there are 26additional nucleotides inserted before the E-box/USF site. The p19promoter, p40 promoter and polyA can also be identified from the AAV-1genome by analogy to known AAV serotypes, which are also highlyconserved.

[0092] Thus, the analysis of AAV terminal repeats of various serotypesshowed that the A and A′ sequence is very much conserved. One of thereasons may be the Rep binding motif (GCTC)₃GCTG [SEQ ID NO: 20]. Thesesequences appear to be essential for AAV DNA replication andsite-specific integration. The same sequence has also been shown to bepreserved in a monkey genome [Samulski, personal communication]. Thefirst 8 nucleotides of the D sequence are also identical in all knownAAV serotypes. This is in accordance with the observation of theSrivastava group that only the first 10 nucleotides are essential forAAV packaging [X. S. Wang et al, J. Virol., 71:3077-3082 (1997); X. S.Wang et al, J. Virol., 71:1140-1146 (1997)]. The function of the rest ofthe D sequences still remain unclear. They may be somehow related totheir tissue specificities. The variation of nucleotide in B and Csequence may also suggest that the secondary structure of the ITRs ismore critical for its biological function, which has been demonstratedin many previous publications.

EXAMPLE 3 Comparison of AAV-1 Sequences

[0093] The nucleotide sequences of AAV-1, obtained as described above,were compared with known AAV sequences, including AAV-2, AAV-4 and AAV-6using DNA Star Megalign. This comparison revealed a stretch of 71identical nucleotides shared by AAV-1, AAV-2 and AAV-6. See, FIGS.1A-1C.

[0094] This comparison further suggested that AAV-6 is a hybrid formedby homologous recombination of AAV-1 and AAV-2. See, FIGS. 3A and 3B.These nucleotides divide the AAV-6 genome into two regions. The 5′ halfof AAV-6 of 522 nucleotides is identical to that of AAV-2 except in 2positions. The 3′ half of AAV-6 including the majority of the rep gene,complete cap gene and 3′ ITR is 98% identical to AAV-1.

[0095] Biologically, such recombination may enable AAV-1 to acquire theability to transmit through the human population. It is also interestingto note that the ITRs of AAV-6 comprise one AAV-1 ITR and one AAV-2 ITR.The replication model of defective parvovirus can maintain this specialarrangement. Studies on AAV integration have shown that a majority ofAAV integrants carries deletions in at least one of the terminalrepeats. These deletions have been shown to be able to be repairedthrough gene conversion using the other intact terminal repeat as atemplate. Therefore, it would be very difficult to maintain AAV-6 as ahomogenous population when an integrated copy of AAV-6 is rescued fromhost cells with helper virus infection. The AAV-6 with two identicalAAV-2 ITRs or two identical AAV-1 ITRs should be the dominant variants.The AAV-6 with two AAV-1 ITRs has been observed by Russell's group[Rutledge, cited above (1998)]. So far there is no report on AAV-6 withtwo AAV-2 ITRs. Acquirement of AAV-2 P5 promoter by AAV-6 may haveexplained that AAV-6 have been isolated from human origin while AAV-1with the same virion has not. The regulation of P5 promoter betweendifferent species of AAV may be different in vivo. This observationsuggests the capsid proteins of AAV were not the only determinants fortissue specificity.

[0096] Although it is clear that AAV-6 is a hybrid of AAV-1 and AAV-2,AAV-6 has already exhibited divergence from either AAV-1 or AAV-2. Thereare two nucleotide differences between AAV-6 and AAV-2 in their first450 nucleotides. There are about 1% differences between AAV-6 and AAV-1in nucleotide levels from nucleotides 522 to the 3′ end. There alsoexists a quite divergent region (nucleotide 4486-4593) between AAV-6 andAAV-1 (FIGS. 1A-1C). This region does not encode any known proteins forAAVs. These differences in nucleotide sequences may suggest that AAV-6and AAV-1 have gone through some evolution since the recombination tookplace. Another possible explanation is that there exists another variantof AAV-1 which has yet to be identified. So far, there is no evidence torule out either possibility. It is still unknown if other hybrids (AAV-2to AAV-4, etc.) existed in nature.

[0097] The coding region of AAV-1 was deduced by comparison with otherknown AAV serotypes. Table 1 illustrates the coding region differencesbetween AAV-1 and AAV-6. The amino acid residues are deduced accordingto AAV-2.

[0098] With reference to the amino acid position of AAV-1, Table 1 liststhe amino acids of AAV-1 which have been changed to the correspondingones of AAV-6. The amino acids of AAV-1 are shown to the left of thearrow. Reference may be made to SEQ ID NO: 5 of the amino acid sequenceof AAV-1 Rep 78 and to SEQ ID NO: 13 for the amino acid sequence ofAAV-1 VP 1. TABLE 1 Coding region variations between AAV-1 and AAV-6Position(s) Amino acids Rep protein (Rep78) 28 S→N 191 Q→H 192 H→D 308E→D Cap protein (VP1) 129 L→F 418 E→D 531 E→K 584 F→L 598 A→V 642 N→H

[0099] It was surprising to see that the sequence of the AAV-1 codingregion is almost identical to that of AAV-6 from position 452 to the endof coding region (99%). The first 508 nucleotides of AAV-6 have beenshown to be identical to those of AAV-2 [Rutledge, cited above (1998)].Since the components of AAV-6 genome seemed to be AAV-2 left ITR—AAV-2p5 promoter—AAV-1 coding region—AAV-1 right ITR, it was concluded thatAAV-6 is a naturally occurred hybrid between AAV-1 and AAV-2.

EXAMPLE 4 Gene Therapy Vector Based on AAV-1

[0100] Recombinant gene transfer vectors based on AAV-1 viruses wereconstructed by the methods described in Example 1. To produce a hybridrecombinant virus with AAV-1 virion and AAV-2 ITR, the AAV-1 transplasmid (pAV1H) and the AAV-2 cis-lacZ plasmid (with AAV-2 ITR) wereused. The AAV-2 ITR was used in this vector in view of its known abilityto direct site-specific integration. Also constructed for use in thisexperiment was an AAV-1 vector carrying the green fluorescent protein(GFP) marker gene under the control of the immediate early promoter ofCMV using pAV1H as the trans plasmid.

[0101] A. rAAV-1 Viruses Transfect Host Cells In Vitro

[0102] 84-31 cells, which are subclones of 293 cells (which expressadenovirus E1a, E1b) which stably express E4/ORF5, were infected withrAAV-1 GFP or rAAV-lacZ. High levels of expression of GFP and lacZ wasdetected in the cultured 84-31 cells. This suggested that rAAV-1 basedvector was very similar to AAV-2 based vectors in ability to infect andexpression levels.

[0103] B. rAAV-1 Viruses Transfect Cells in Vivo

[0104] The performance of AAV-1 based vectors was also tested in vivo.The rAAV-1 CMV-α1AT virus was constructed as follows. The EcoRI fragmentof pAT85 (ATCC) containing human α1-antitrypsin (α1AT) cDNA fragment wasblunted and cloned into PCR (Promega) at a SmaI site to obtain PCR-α1AT.The CMV promoter was cloned into PCR-α1AT at the XbaI site. The Alb-α1ATexpression cassette was removed by XhoI and ClaI and cloned into pAV1Hat the XbaI site. This vector plasmid was used to generateAAV-1-CMV-α1AT virus used in the experiment described below.

[0105] For screening human antibodies against AAV, purified AAV virus islysed with Ripa buffer (10 mM Tris pH 8.2, 1% Triton X-100, 1% SDS, 0.15M NaCl) and separated in 10% SDS-PAGE gel. The heat inactivated humanserum was used at a 1 to 1000 dilution in this assay. The rAAV-1CMV-α1AT viruses were injected into Rag-1 mice through tail veininjection at different dosages. The concentration of humanα1-antitrypsin in mouse serum was measured using ELISA. The coatingantibody is rabbit anti-human human α1-antitrypsin (Sigma). Thegoat-antihuman α1-antitrypsin (Sigma) was used as the primary detectionantibodies. The sensitivity of this assay is around 0.3 ng/ml to 30ng/ml. The expression of human α-antitrypsin in mouse blood can bedetected in a very encouraging level. This result is shown in Table 2.TABLE 2 Human Antitrypsin Expressed in Mouse Liver Amount of virusinjected Week 2 (ng/ml) Week 4 (ng/ml)   2 × 10¹⁰ genomes 214.2 171.4  1 × 10¹⁰ genomes 117.8 109.8   5 × 10¹⁰ genomes 64.5 67.8 2.5 × 10¹⁰genomes 30.9 58.4

[0106] rAAV-1 CMV-lacZ viruses were also injected into the muscle ofC57BL6 mice and similar results were obtained. Collectively, theseresults suggested that AAV-1 based vector would be appropriate for bothliver and muscle gene delivery.

EXAMPLE 5 Neutralizing Antibodies Against AAV-1

[0107] Simple and quantitative assays for neutralizing antibodies (NAB)to AAV-1 and AAV-2 were developed with recombinant vectors. A total of33 rhesus monkeys and 77 normal human subjects were screened.

[0108] 1. Nonhuman Primates

[0109] Wild-caught juvenile rhesus monkeys were purchased from Covance(Alice, Tex.) and LABS of Virginia (Yemassee, S.C.) and kept in fullquarantine. The monkeys weighed approximately 3 to 4 kg. The nonhumanprimates used in the Institute for Human Gene Therapy research programare purposefully bred in the United States from specific-pathogen-freeclosed colonies. All vendors are US Department of Agriculture class Adealers. The rhesus macaques are therefore not infected with importantsimian pathogens, including the tuberculosis agent, major simianlentiviruses (simian immunodeficiency virus and simian retroviruses),and cercopithecine herpesvirus. The animals are also free of internaland external parasites. The excellent health status of these premiumanimals minimized the potential for extraneous variables. For thisstudy, serum was obtained from monkeys prior to initiation of anyprotocol.

[0110] NAB titers were analyzed by assessing the ability of serumantibody to inhibit the transduction of reporter virus expressing greenfluorescent protein (GFP) (AAV1-GFP or AAV2-GFP) into 84-31 cells.Various dilutions of antibodies preincubated with reporter virus for 1hour at 37° C. were added to 90% confluent cell cultures. Cells wereincubated for 48 hours and the expression of green fluorescent proteinwas measured by Fluorolmaging (Molecular Dynamics). NAB titers werecalculated as the highest dilution at which 50% of the cells stainedgreen.

[0111] Analysis of NAB in rhesus monkeys showed that 61% of animalstested positive for AAV-1; a minority (24%) has NAB to AAV-2. Overone-third of animals had antibodies to AAV-1 but not AAV-2 (i.e., weremonospecific for AAV-1), whereas no animals were positive for AAV-2without reacting to AAV-1. These data support the hypothesis that AAV-1is endemic in rhesus monkeys. The presence of true AAV-2 infections inthis group of nonhuman primates is less clear, since cross-neutralizingactivity of an AAV-1 response to AAV-2 can not be ruled out. It isinteresting that there is a linear relationship between AAV-2 NAB andAAV-1 NAB in animals that had both.

[0112] 2. Humans

[0113] For these neutralization antibody assays, human serum sampleswere incubated at 56° C. for 30 min to inactivate complement and thendiluted in DMEM. The virus (rAAV or rAd with either lacZ or GFP) wasthen mixed with each serum dilution (20×, 400×, 2000×, 4000×, etc.) andincubated for 1 hour at 37° C. before applied to 90% confluent culturesof 84-31 cells (for AAV) or Hela cells (for adenovirus) in 96-wellplates. After 60 minutes of incubation at culture condition, 100 μladditional media containing 20% FCS was added to make final culturemedia containing 10% FCS.

[0114] The result is summarized in Table 3. TABLE 3 Adenovirus AAV-1AAV-2 # of samples Percentage − − − 41 53.2% + − − 16 20.8% − + − 0 0.0% − − + 2  2.6% − + + 2  2.6% + − + 3  3.9% + + − 0  0.0% + + + 1316.9% Total 77  100%

[0115] The human neutralizing antibodies against these three virusesseemed to be unrelated since the existence of neutralizing antibodiesagainst AAV are not indications for antibodies against adenovirus.However, AAV requires adenovirus as helper virus, in most of the cases,the neutralizing antibodies against AAV correlated with the existence ofneutralizing antibodies to adenovirus. Among the 77 human serum samplesscreened, 41% of the samples can neutralize the infectivity ofrecombinant adenovirus based on Ad5. 15/77 (19%) of serum samples canneutralize the transduction of rAAV-1 while 20/77 (20%) of the samplesinhibit rAAV-2 transduction at 1 to 80 dilutions or higher. All serumsamples positive in neutralizing antibodies for AAV-1 in are alsopositive for AAV-2. However, there are five (6%) rAAV-2 positive samplesthat failed to neutralize rAAV-1. In samples that are positive forneutralizing antibodies, the titer of antibodies also varied in thepositive ones. The results from screening human sera for antibodiesagainst AAVs supported the conclusion that AAV-1 presents the sameepitome as that of AAV-2 to interact with cellular receptors since AAV-1neutralizing human serums can also decrease the infectivity of AAV-2.However, the profile of neutralizing antibodies for these AAVs is notidentical, there are additional specific receptors for each AAVserotype.

EXAMPLE 6 Recombinant AAV Viruses Exhibit Tissue Tropism

[0116] The recombinant AAV-1 vectors of the invention and therecombinant AAV-2 vectors [containing the gene encoding humanα1-antitrypsin (α1AT) or murine erythropoietin (Epo) from acytomegalovirus-enhanced β-actin promoter (CB)] were evaluated in adirect comparison to equivalent copies of AAV-2 vectors containing thesame vector genes.

[0117] Recombinant viruses with AAV-1 capsids were constructed using thetechniques in Example 1. To make rAAV with AAV-1 virions, pAV1H or p5E18 (2/1) was used as the trans plasmid to provide Rep and Capfunctions. For the generation of the rAAV based on AAV-2, p5E 18(2/2)was used as the trans plasmid, since it greatly improved the rAAV yield.[Early experiments indicated similar in vivo performances of AAV-1vectors produced with pAV1H and p5E19 (2/1). All subsequent studies usedAAV-1 vectors derived from p5E18(2/1) because of the increased yield.]

[0118] Equivalent stocks of the AAV-1 and AAV-2 vectors were injectedintramuscularly (5×10¹⁰ genomes) or liver via the portal circulation(1×10¹¹ genomes) into immunodeficient mice, and the animals (fourgroups) were analyzed on day 30 for expression of transgene. See, FIGS.4A and 4B.

[0119] AAV-2 vectors consistently produced 10- to 50-fold more serumerythropoietin or al-antitrypsin when injected into liver compared tomuscle. (However, the AAV-1-delivered genes did achieve acceptableexpression levels in the liver.) This result was very different fromthat for AAV-1 vectors, with which muscle expression was equivalent toor greater than liver expression. In fact, AAV-1 outperformed AAV-2 inmuscle when equivalent titers based on genomes were administered.

EXAMPLE 7 Gene Delivery Via rAAV-1

[0120] C57BL/6 mice (6- to 8-week old males, Jackson Laboratories) wereanalyzed for AAV mediated gene transfer to liver following intrasplenicinjection of vector (i.e., targeted to liver). A total of 10¹¹ genomeequivalents of rAAV-1 or rAAV-2 vector were injected into thecirculation in 100 μl buffered saline. The first vector contained eitheran AAV-1 capsid or an AAV-2 capsid and expressed α1AT under the controlof the chicken β-actin (CB) promoter. Day 28 sera were analyzed forantibodies against AAV-1 or AAV-2 and serum α1AT levels were checked.Animals were then injected with an AAV-1 or AAV-2 construct expressingerythropoietin (Epo, also under the control of the CB promoter). Onemonth later sera was analyzed for serum levels of Epo. The followinggroups were analyzed (FIGS. 5A-5D).

[0121] In Group 1, vector 1 was AAV-2 expressing α1AT and vector 2 wasAAV-2 expressing Epo. Animals generated antibodies against AAV-2following the first vector administration which prevented thereadministration of the AAV-2 based vector. There was no evidence forcross-neutralizing the antibody to AAV-1.

[0122] In Group 2, vector 1 was AAV-1 expressing α1AT while vector 2 wasAAV-1 expressing Epo. The first vector administration did result insignificant α1AT expression at one month associated with antibodies toneutralizing antibodies to AAV-1. The animals were not successfullyreadministered with the AAV-1 Epo expressing construct.

[0123] In Group 3, the effectiveness of an AAV-2 vector expressing Epoinjected into a naive animal was measured. The animals were injectedwith PBS and injected with AAV-2 Epo vector at day 28 and analyzed forEpo expression one month later. The neutralizing antibodies wereevaluated at day 28 so we did not expect to see anything since theyreceived PBS with the first vector injection. This shows that in naiveanimals AAV-2 is very efficient at transferring the Epo gene asdemonstrated by high level of serum Epo one month later.

[0124] Group 4 was an experiment similar to Group 3 in which the animalsoriginally received PBS for vector 1 and then the AAV-1 expressing Epoconstruct 28 days later. At the time of vector injection, thereobviously were no antibodies to either AAV-1 or AAV-2. The AAV-1 basedvector was capable of generating significant expression of Epo whenmeasured one month later.

[0125] Group 5 is a cross-over experiment where the initial vector isAAV-2 expressing α1AT followed by the AAV-1 construct expressing Epo.The animals, as expected, were efficiently infected with the AAV-2vector expressing α1AT as shown by high levels of the protein in bloodat 28 days. This was associated with significant neutralizing antibodiesto AAV-2. Importantly, the animals were successfully administered AAV-1following the AAV-2 vector as shown by the presence of Epo in serum 28days following the second vector administration. At the time of thisvector administration, there was high level AAV-2 neutralizingantibodies and very low cross-reaction to AAV-1. The level of Epo wasslightly diminished possibly due to a small amount of cross-reactivity.Group 6 was the opposite cross-over experiment in which the initialvector was AAV-1 based, whereas the second experiment was AAV-2 based.The AAV-1 vector did lead to significant gene expression of α1AT, whichalso resulted in high level AAV-1 neutralizing antibody. The animalswere very efficiently administered AAV-2 following the initial AAV-1vector as evidenced by high level Epo.

[0126] A substantially identical experiment was performed in muscle inwhich 5×10¹⁰ genomes were injected into the tibialis anterior of C57BL/6mice as a model for muscle directed gene therapy. The results areillustrated in FIGS. 6A-6D and are essentially the same as for liver.

[0127] In summary, this experiment demonstrates the utility of using anAAV-1 vector in patients who have pre-existing antibodies to AAV-2 orwho had initially received an AAV-2 vector and need readministration.

EXAMPLE 8 Construction of Recombinant Viruses Containing AAV-1 ITRs

[0128] This example illustrates the construction of recombinant AAVvectors which contain AAV-1 ITRs of the invention.

[0129] An AAV-1 cis plasmid is constructed as follows. A 160 bp Xho-NruIAAV-1 fragment containing the AAV-1 5′ ITR is obtained from pAV1-BL.pAV1-BL was generated as described in Example 1. The Xho-NruI fragmentis then cloned into a second pAV1-BL plasmid at an XbaI site to providethe plasmid with two AAV-1 ITRs. The desired transgene is then clonedinto the modified pAV-1 BL at the NruI and BamHI site, which is locatedbetween the AAV-1 ITR sequences. The resulting AAV-1 cis plasmidcontains AAV-1 ITRs flanking the transgene and lacks functional AAV-1rep and cap.

[0130] Recombinant AAV is produced by simultaneously transfecting threeplasmids into 293 cells. These include the AAV-1 cis plasmid describedabove; a trans plasmid which provides AAV rep/cap functions and lacksAAV ITRs; and a plasmid providing adenovirus helper functions. The repand/or cap functions may be provided in trans by AAV-1 or another AAVserotype, depending on the immunity profile of the intended recipient.Alternatively, the rep or cap functions may be provided in cis by AAV-1or another serotype, again depending on the patient's immunity profile.

[0131] In a typical cotransfection, 50 μg of DNA (cis:trans:helper atratios of 1:1:2, respectively) is transfected onto a 15 cm tissueculture dish. Cells are harvested 96 hours post transfection, sonicatedand treated with 0.5% sodium deoxycholate (37° for 10 min). Cell lysatesare then subjected to 2-3 rounds of ultracentrifugation in a cesiumgradient. Peak fractions containing rAAV are collected, pooled anddialyzed against PBS. A typical yield is 1×10¹³ genomes/10⁹ cells.

[0132] Using this method, one recombinant virus construct is preparedwhich contains the AAV-1 ITRs flanking the transgene, with an AAV-1capsid. Another recombinant virus construct is prepared with containsthe AAV-1 ITRs flanking the transgene, with an AAV-2 capsid.

[0133] All publications cited in this specification are incorporatedherein by reference. While the invention has been described withreference to a particularly preferred embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the claims.

1 20 1 4718 DNA AAV-1 CDS (335)..(2206) 1 ttgcccactc cctctctgcgcgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc 60 agacggcaga gctctgctctgccggcccca ccgagcgagc gagcgcgcag agagggagtg 120 ggcaactcca tcactaggggtaatcgcgaa gcgcctccca cgctgccgcg tcagcgctga 180 cgtaaattac gtcataggggagtggtcctg tattagctgt cacgtgagtg cttttgcgac 240 attttgcgac accacgtggccatttagggt atatatggcc gagtgagcga gcaggatctc 300 cattttgacc gcgaaatttgaacgagcagc agcc atg ccg ggc ttc tac gag atc 355 Met Pro Gly Phe Tyr GluIle 1 5 gtg atc aag gtg ccg agc gac ctg gac gag cac ctg ccg ggc att tct403 Val Ile Lys Val Pro Ser Asp Leu Asp Glu His Leu Pro Gly Ile Ser 1015 20 gac tcg ttt gtg agc tgg gtg gcc gag aag gaa tgg gag ctg ccc ccg451 Asp Ser Phe Val Ser Trp Val Ala Glu Lys Glu Trp Glu Leu Pro Pro 2530 35 gat tct gac atg gat ctg aat ctg att gag cag gca ccc ctg acc gtg499 Asp Ser Asp Met Asp Leu Asn Leu Ile Glu Gln Ala Pro Leu Thr Val 4045 50 55 gcc gag aag ctg cag cgc gac ttc ctg gtc caa tgg cgc cgc gtg agt547 Ala Glu Lys Leu Gln Arg Asp Phe Leu Val Gln Trp Arg Arg Val Ser 6065 70 aag gcc ccg gag gcc ctc ttc ttt gtt cag ttc gag aag ggc gag tcc595 Lys Ala Pro Glu Ala Leu Phe Phe Val Gln Phe Glu Lys Gly Glu Ser 7580 85 tac ttc cac ctc cat att ctg gtg gag acc acg ggg gtc aaa tcc atg643 Tyr Phe His Leu His Ile Leu Val Glu Thr Thr Gly Val Lys Ser Met 9095 100 gtg ctg ggc cgc ttc ctg agt cag att agg gac aag ctg gtg cag acc691 Val Leu Gly Arg Phe Leu Ser Gln Ile Arg Asp Lys Leu Val Gln Thr 105110 115 atc tac cgc ggg atc gag ccg acc ctg ccc aac tgg ttc gcg gtg acc739 Ile Tyr Arg Gly Ile Glu Pro Thr Leu Pro Asn Trp Phe Ala Val Thr 120125 130 135 aag acg cgt aat ggc gcc gga ggg ggg aac aag gtg gtg gac gagtgc 787 Lys Thr Arg Asn Gly Ala Gly Gly Gly Asn Lys Val Val Asp Glu Cys140 145 150 tac atc ccc aac tac ctc ctg ccc aag act cag ccc gag ctg cagtgg 835 Tyr Ile Pro Asn Tyr Leu Leu Pro Lys Thr Gln Pro Glu Leu Gln Trp155 160 165 gcg tgg act aac atg gag gag tat ata agc gcc tgt ttg aac ctggcc 883 Ala Trp Thr Asn Met Glu Glu Tyr Ile Ser Ala Cys Leu Asn Leu Ala170 175 180 gag cgc aaa cgg ctc gtg gcg cag cac ctg acc cac gtc agc cagacc 931 Glu Arg Lys Arg Leu Val Ala Gln His Leu Thr His Val Ser Gln Thr185 190 195 cag gag cag aac aag gag aat ctg aac ccc aat tct gac gcg cctgtc 979 Gln Glu Gln Asn Lys Glu Asn Leu Asn Pro Asn Ser Asp Ala Pro Val200 205 210 215 atc cgg tca aaa acc tcc gcg cgc tac atg gag ctg gtc gggtgg ctg 1027 Ile Arg Ser Lys Thr Ser Ala Arg Tyr Met Glu Leu Val Gly TrpLeu 220 225 230 gtg gac cgg ggc atc acc tcc gag aag cag tgg atc cag gaggac cag 1075 Val Asp Arg Gly Ile Thr Ser Glu Lys Gln Trp Ile Gln Glu AspGln 235 240 245 gcc tcg tac atc tcc ttc aac gcc gct tcc aac tcg cgg tcccag atc 1123 Ala Ser Tyr Ile Ser Phe Asn Ala Ala Ser Asn Ser Arg Ser GlnIle 250 255 260 aag gcc gct ctg gac aat gcc ggc aag atc atg gcg ctg accaaa tcc 1171 Lys Ala Ala Leu Asp Asn Ala Gly Lys Ile Met Ala Leu Thr LysSer 265 270 275 gcg ccc gac tac ctg gta ggc ccc gct ccg ccc gcg gac attaaa acc 1219 Ala Pro Asp Tyr Leu Val Gly Pro Ala Pro Pro Ala Asp Ile LysThr 280 285 290 295 aac cgc atc tac cgc atc ctg gag ctg aac ggc tac gaacct gcc tac 1267 Asn Arg Ile Tyr Arg Ile Leu Glu Leu Asn Gly Tyr Glu ProAla Tyr 300 305 310 gcc ggc tcc gtc ttt ctc ggc tgg gcc cag aaa agg ttcggg aag cgc 1315 Ala Gly Ser Val Phe Leu Gly Trp Ala Gln Lys Arg Phe GlyLys Arg 315 320 325 aac acc atc tgg ctg ttt ggg ccg gcc acc acg ggc aagacc aac atc 1363 Asn Thr Ile Trp Leu Phe Gly Pro Ala Thr Thr Gly Lys ThrAsn Ile 330 335 340 gcg gaa gcc atc gcc cac gcc gtg ccc ttc tac ggc tgcgtc aac tgg 1411 Ala Glu Ala Ile Ala His Ala Val Pro Phe Tyr Gly Cys ValAsn Trp 345 350 355 acc aat gag aac ttt ccc ttc aat gat tgc gtc gac aagatg gtg atc 1459 Thr Asn Glu Asn Phe Pro Phe Asn Asp Cys Val Asp Lys MetVal Ile 360 365 370 375 tgg tgg gag gag ggc aag atg acg gcc aag gtc gtggag tcc gcc aag 1507 Trp Trp Glu Glu Gly Lys Met Thr Ala Lys Val Val GluSer Ala Lys 380 385 390 gcc att ctc ggc ggc agc aag gtg cgc gtg gac caaaag tgc aag tcg 1555 Ala Ile Leu Gly Gly Ser Lys Val Arg Val Asp Gln LysCys Lys Ser 395 400 405 tcc gcc cag atc gac ccc acc ccc gtg atc gtc acctcc aac acc aac 1603 Ser Ala Gln Ile Asp Pro Thr Pro Val Ile Val Thr SerAsn Thr Asn 410 415 420 atg tgc gcc gtg att gac ggg aac agc acc acc ttcgag cac cag cag 1651 Met Cys Ala Val Ile Asp Gly Asn Ser Thr Thr Phe GluHis Gln Gln 425 430 435 ccg ttg cag gac cgg atg ttc aaa ttt gaa ctc acccgc cgt ctg gag 1699 Pro Leu Gln Asp Arg Met Phe Lys Phe Glu Leu Thr ArgArg Leu Glu 440 445 450 455 cat gac ttt ggc aag gtg aca aag cag gaa gtcaaa gag ttc ttc cgc 1747 His Asp Phe Gly Lys Val Thr Lys Gln Glu Val LysGlu Phe Phe Arg 460 465 470 tgg gcg cag gat cac gtg acc gag gtg gcg catgag ttc tac gtc aga 1795 Trp Ala Gln Asp His Val Thr Glu Val Ala His GluPhe Tyr Val Arg 475 480 485 aag ggt gga gcc aac aaa aga ccc gcc ccc gatgac gcg gat aaa agc 1843 Lys Gly Gly Ala Asn Lys Arg Pro Ala Pro Asp AspAla Asp Lys Ser 490 495 500 gag ccc aag cgg gcc tgc ccc tca gtc gcg gatcca tcg acg tca gac 1891 Glu Pro Lys Arg Ala Cys Pro Ser Val Ala Asp ProSer Thr Ser Asp 505 510 515 gcg gaa gga gct ccg gtg gac ttt gcc gac aggtac caa aac aaa tgt 1939 Ala Glu Gly Ala Pro Val Asp Phe Ala Asp Arg TyrGln Asn Lys Cys 520 525 530 535 tct cgt cac gcg ggc atg ctt cag atg ctgttt ccc tgc aag aca tgc 1987 Ser Arg His Ala Gly Met Leu Gln Met Leu PhePro Cys Lys Thr Cys 540 545 550 gag aga atg aat cag aat ttc aac att tgcttc acg cac ggg acg aga 2035 Glu Arg Met Asn Gln Asn Phe Asn Ile Cys PheThr His Gly Thr Arg 555 560 565 gac tgt tca gag tgc ttc ccc ggc gtg tcagaa tct caa ccg gtc gtc 2083 Asp Cys Ser Glu Cys Phe Pro Gly Val Ser GluSer Gln Pro Val Val 570 575 580 aga aag agg acg tat cgg aaa ctc tgt gccatt cat cat ctg ctg ggg 2131 Arg Lys Arg Thr Tyr Arg Lys Leu Cys Ala IleHis His Leu Leu Gly 585 590 595 cgg gct ccc gag att gct tgc tcg gcc tgcgat ctg gtc aac gtg gac 2179 Arg Ala Pro Glu Ile Ala Cys Ser Ala Cys AspLeu Val Asn Val Asp 600 605 610 615 ctg gat gac tgt gtt tct gag caa taaatgacttaaa ccaggt atg gct gcc 2231 Leu Asp Asp Cys Val Ser Glu Gln MetAla Ala 620 625 gat ggt tat ctt cca gat tgg ctc gag gac aac ctc tct gagggc att 2279 Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu GlyIle 630 635 640 cgc gag tgg tgg gac ttg aaa cct gga gcc ccg aag ccc aaagcc aac 2327 Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro Lys AlaAsn 645 650 655 cag caa aag cag gac gac ggc cgg ggt ctg gtg ctt cct ggctac aag 2375 Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro Gly TyrLys 660 665 670 tac ctc gga ccc ttc aac gga ctc gac aag ggg gag ccc gtcaac gcg 2423 Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro Val AsnAla 675 680 685 690 gcg gac gca gcg gcc ctc gag cac gac aag gcc tac gaccag cag ctc 2471 Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp GlnGln Leu 695 700 705 aaa gcg ggt gac aat ccg tac ctg cgg tat aac cac gccgac gcc gag 2519 Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala AspAla Glu 710 715 720 ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggcaac ctc ggg 2567 Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly AsnLeu Gly 725 730 735 cga gca gtc ttc cag gcc aag aag cgg gtt ctc gaa cctctc ggt ctg 2615 Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro LeuGly Leu 740 745 750 gtt gag gaa ggc gct aag acg gct cct gga aag aaa cgtccg gta gag 2663 Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg ProVal Glu 755 760 765 770 cag tcg cca caa gag cca gac tcc tcc tcg ggc atcggc aag aca ggc 2711 Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile GlyLys Thr Gly 775 780 785 cag cag ccc gct aaa aag aga ctc aat ttt ggt cagact ggc gac tca 2759 Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln ThrGly Asp Ser 790 795 800 gag tca gtc ccc gat cca caa cct ctc gga gaa cctcca gca acc ccc 2807 Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro ProAla Thr Pro 805 810 815 gct gct gtg gga cct act aca atg gct tca ggc ggtggc gca cca atg 2855 Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly GlyAla Pro Met 820 825 830 gca gac aat aac gaa ggc gcc gac gga gtg ggt aatgcc tca gga aat 2903 Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn AlaSer Gly Asn 835 840 845 850 tgg cat tgc gat tcc aca tgg ctg ggc gac agagtc atc acc acc agc 2951 Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg ValIle Thr Thr Ser 855 860 865 acc cgc acc tgg gcc ttg ccc acc tac aat aaccac ctc tac aag caa 2999 Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn HisLeu Tyr Lys Gln 870 875 880 atc tcc agt gct tca acg ggg gcc agc aac gacaac cac tac ttc ggc 3047 Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp AsnHis Tyr Phe Gly 885 890 895 tac agc acc ccc tgg ggg tat ttt gat ttc aacaga ttc cac tgc cac 3095 Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn ArgPhe His Cys His 900 905 910 ttt tca cca cgt gac tgg cag cga ctc atc aacaac aat tgg gga ttc 3143 Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn AsnAsn Trp Gly Phe 915 920 925 930 cgg ccc aag aga ctc aac ttc aaa ctc ttcaac atc caa gtc aag gag 3191 Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe AsnIle Gln Val Lys Glu 935 940 945 gtc acg acg aat gat ggc gtc aca acc atcgct aat aac ctt acc agc 3239 Val Thr Thr Asn Asp Gly Val Thr Thr Ile AlaAsn Asn Leu Thr Ser 950 955 960 acg gtt caa gtc ttc tcg gac tcg gag taccag ctt ccg tac gtc ctc 3287 Thr Val Gln Val Phe Ser Asp Ser Glu Tyr GlnLeu Pro Tyr Val Leu 965 970 975 ggc tct gcg cac cag ggc tgc ctc cct ccgttc ccg gcg gac gtg ttc 3335 Gly Ser Ala His Gln Gly Cys Leu Pro Pro PhePro Ala Asp Val Phe 980 985 990 atg att ccg caa tac ggc tac ctg acg ctcaac aat ggc agc caa 3380 Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn AsnGly Ser Gln 995 1000 1005 gcc gtg gga cgt tca tcc ttt tac tgc ctg gaatat ttc cct tct 3425 Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr PhePro Ser 1010 1015 1020 cag atg ctg aga acg ggc aac aac ttt acc ttc agctac acc ttt 3470 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr ThrPhe 1025 1030 1035 gag gaa gtg cct ttc cac agc agc tac gcg cac agc cagagc ctg 3515 Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu1040 1045 1050 gac cgg ctg atg aat cct ctc atc gac caa tac ctg tat tacctg 3560 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu1055 1060 1065 aac aga act caa aat cag tcc gga agt gcc caa aac aag gacttg 3605 Asn Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu1070 1075 1080 ctg ttt agc cgt ggg tct cca gct ggc atg tct gtt cag cccaaa 3650 Leu Phe Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys1085 1090 1095 aac tgg cta cct gga ccc tgt tat cgg cag cag cgc gtt tctaaa 3695 Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys1100 1105 1110 aca aaa aca gac aac aac aac agc aat ttt acc tgg act ggtgct 3740 Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala1115 1120 1125 tca aaa tat aac ctc aat ggg cgt gaa tcc atc atc aac cctggc 3785 Ser Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly1130 1135 1140 act gct atg gcc tca cac aaa gac gac gaa gac aag ttc tttccc 3830 Thr Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro1145 1150 1155 atg agc ggt gtc atg att ttt gga aaa gag agc gcc gga gcttca 3875 Met Ser Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser1160 1165 1170 aac act gca ttg gac aat gtc atg att aca gac gaa gag gaaatt 3920 Asn Thr Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile1175 1180 1185 aaa gcc act aac cct gtg gcc acc gaa aga ttt ggg acc gtggca 3965 Lys Ala Thr Asn Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala1190 1195 1200 gtc aat ttc cag agc agc agc aca gac cct gcg acc gga gatgtg 4010 Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala Thr Gly Asp Val1205 1210 1215 cat gct atg gga gca tta cct ggc atg gtg tgg caa gat agagac 4055 His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp Arg Asp1220 1225 1230 gtg tac ctg cag ggt ccc att tgg gcc aaa att cct cac acagat 4100 Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp1235 1240 1245 gga cac ttt cac ccg tct cct ctt atg ggc ggc ttt gga ctcaag 4145 Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys1250 1255 1260 aac ccg cct cct cag atc ctc atc aaa aac acg cct gtt cctgcg 4190 Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala1265 1270 1275 aat cct ccg gcg gag ttt tca gct aca aag ttt gct tca ttcatc 4235 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile1280 1285 1290 acc caa tac tcc aca gga caa gtg agt gtg gaa att gaa tgggag 4280 Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu1295 1300 1305 ctg cag aaa gaa aac agc aag cgc tgg aat ccc gaa gtg cagtac 4325 Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr1310 1315 1320 aca tcc aat tat gca aaa tct gcc aac gtt gat ttt act gtggac 4370 Thr Ser Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp1325 1330 1335 aac aat gga ctt tat act gag cct cgc ccc att ggc acc cgttac 4415 Asn Asn Gly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr1340 1345 1350 ctt acc cgt ccc ctg taattacgtg ttaatcaata aaccggttgattcgtttcag 4470 Leu Thr Arg Pro Leu 1355 ttgaactttg gtctcctgtccttcttatct tatcggttac catggttata gcttacacat 4530 taactgcttg gttgcgcttcgcgataaaag acttacgtca tcgggttacc cctagtgatg 4590 gagttgccca ctccctctctgcgcgctcgc tcgctcggtg gggcctgcgg accaaaggtc 4650 cgcagacggc agagctctgctctgccggcc ccaccgagcg agcgagcgcg cagagaggga 4710 gtgggcaa 4718 2 623 PRTAAV-1 2 Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp Leu Asp1 5 10 15 Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Ser Trp Val AlaGlu 20 25 30 Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp Leu Asn LeuIle 35 40 45 Glu Gln Ala Pro Leu Thr Val Ala Glu Lys Leu Gln Arg Asp PheLeu 50 55 60 Val Gln Trp Arg Arg Val Ser Lys Ala Pro Glu Ala Leu Phe PheVal 65 70 75 80 Gln Phe Glu Lys Gly Glu Ser Tyr Phe His Leu His Ile LeuVal Glu 85 90 95 Thr Thr Gly Val Lys Ser Met Val Leu Gly Arg Phe Leu SerGln Ile 100 105 110 Arg Asp Lys Leu Val Gln Thr Ile Tyr Arg Gly Ile GluPro Thr Leu 115 120 125 Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asn GlyAla Gly Gly Gly 130 135 140 Asn Lys Val Val Asp Glu Cys Tyr Ile Pro AsnTyr Leu Leu Pro Lys 145 150 155 160 Thr Gln Pro Glu Leu Gln Trp Ala TrpThr Asn Met Glu Glu Tyr Ile 165 170 175 Ser Ala Cys Leu Asn Leu Ala GluArg Lys Arg Leu Val Ala Gln His 180 185 190 Leu Thr His Val Ser Gln ThrGln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 Pro Asn Ser Asp Ala ProVal Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215 220 Met Glu Leu Val GlyTrp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 Gln Trp IleGln Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255 Ser AsnSer Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265 270 IleMet Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 275 280 285Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 290 295300 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 305310 315 320 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly ProAla 325 330 335 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His AlaVal Pro 340 345 350 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe ProPhe Asn Asp 355 360 365 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu GlyLys Met Thr Ala 370 375 380 Lys Val Val Glu Ser Ala Lys Ala Ile Leu GlyGly Ser Lys Val Arg 385 390 395 400 Val Asp Gln Lys Cys Lys Ser Ser AlaGln Ile Asp Pro Thr Pro Val 405 410 415 Ile Val Thr Ser Asn Thr Asn MetCys Ala Val Ile Asp Gly Asn Ser 420 425 430 Thr Thr Phe Glu His Gln GlnPro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 Glu Leu Thr Arg Arg LeuGlu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460 Glu Val Lys Glu PhePhe Arg Trp Ala Gln Asp His Val Thr Glu Val 465 470 475 480 Ala His GluPhe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495 Pro AspAsp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505 510 AlaAsp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 515 520 525Asp Arg Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu Gln Met 530 535540 Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn Phe Asn Ile 545550 555 560 Cys Phe Thr His Gly Thr Arg Asp Cys Ser Glu Cys Phe Pro GlyVal 565 570 575 Ser Glu Ser Gln Pro Val Val Arg Lys Arg Thr Tyr Arg LysLeu Cys 580 585 590 Ala Ile His His Leu Leu Gly Arg Ala Pro Glu Ile AlaCys Ser Ala 595 600 605 Cys Asp Leu Val Asn Val Asp Leu Asp Asp Cys ValSer Glu Gln 610 615 620 3 736 PRT AAV-1 3 Met Ala Ala Asp Gly Tyr LeuPro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu TrpTrp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln LysGln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu GlyPro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp AlaAla Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys AlaGly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe GlnGlu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly ArgAla Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly LeuVal Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro ValGlu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly AsnAla 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp ArgVal Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr TyrAsn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly AlaSer Asn Asp Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly TyrPhe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp TrpGln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg LeuAsn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr ThrAsn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr ValGln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu GlySer Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val PheMet Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser GlnAla Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410415 Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg435 440 445 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu PheSer 450 455 460 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn TrpLeu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys ThrLys Thr Asp Asn 485 490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala SerLys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly ThrAla Met Ala Ser His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro MetSer Gly Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly Ala Ser AsnThr Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu IleLys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 Phe Gly Thr Val AlaVal Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590 Thr Gly Asp ValHis Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg AspVal Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr AspGly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650655 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr SerAsn 690 695 700 Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn AsnGly Leu 705 710 715 720 Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr LeuThr Arg Pro Leu 725 730 735 4 1872 DNA AAV-1 CDS (1)..(1869) 4 atg ccgggc ttc tac gag atc gtg atc aag gtg ccg agc gac ctg gac 48 Met Pro GlyPhe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp Leu Asp 1 5 10 15 gag cacctg ccg ggc att tct gac tcg ttt gtg agc tgg gtg gcc gag 96 Glu His LeuPro Gly Ile Ser Asp Ser Phe Val Ser Trp Val Ala Glu 20 25 30 aag gaa tgggag ctg ccc ccg gat tct gac atg gat ctg aat ctg att 144 Lys Glu Trp GluLeu Pro Pro Asp Ser Asp Met Asp Leu Asn Leu Ile 35 40 45 gag cag gca cccctg acc gtg gcc gag aag ctg cag cgc gac ttc ctg 192 Glu Gln Ala Pro LeuThr Val Ala Glu Lys Leu Gln Arg Asp Phe Leu 50 55 60 gtc caa tgg cgc cgcgtg agt aag gcc ccg gag gcc ctc ttc ttt gtt 240 Val Gln Trp Arg Arg ValSer Lys Ala Pro Glu Ala Leu Phe Phe Val 65 70 75 80 cag ttc gag aag ggcgag tcc tac ttc cac ctc cat att ctg gtg gag 288 Gln Phe Glu Lys Gly GluSer Tyr Phe His Leu His Ile Leu Val Glu 85 90 95 acc acg ggg gtc aaa tccatg gtg ctg ggc cgc ttc ctg agt cag att 336 Thr Thr Gly Val Lys Ser MetVal Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110 agg gac aag ctg gtg cagacc atc tac cgc ggg atc gag ccg acc ctg 384 Arg Asp Lys Leu Val Gln ThrIle Tyr Arg Gly Ile Glu Pro Thr Leu 115 120 125 ccc aac tgg ttc gcg gtgacc aag acg cgt aat ggc gcc gga ggg ggg 432 Pro Asn Trp Phe Ala Val ThrLys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140 aac aag gtg gtg gac gagtgc tac atc ccc aac tac ctc ctg ccc aag 480 Asn Lys Val Val Asp Glu CysTyr Ile Pro Asn Tyr Leu Leu Pro Lys 145 150 155 160 act cag ccc gag ctgcag tgg gcg tgg act aac atg gag gag tat ata 528 Thr Gln Pro Glu Leu GlnTrp Ala Trp Thr Asn Met Glu Glu Tyr Ile 165 170 175 agc gcc tgt ttg aacctg gcc gag cgc aaa cgg ctc gtg gcg cag cac 576 Ser Ala Cys Leu Asn LeuAla Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 ctg acc cac gtc agccag acc cag gag cag aac aag gag aat ctg aac 624 Leu Thr His Val Ser GlnThr Gln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 ccc aat tct gac gcgcct gtc atc cgg tca aaa acc tcc gcg cgc tac 672 Pro Asn Ser Asp Ala ProVal Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215 220 atg gag ctg gtc gggtgg ctg gtg gac cgg ggc atc acc tcc gag aag 720 Met Glu Leu Val Gly TrpLeu Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 cag tgg atc caggag gac cag gcc tcg tac atc tcc ttc aac gcc gct 768 Gln Trp Ile Gln GluAsp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255 tcc aac tcg cggtcc cag atc aag gcc gct ctg gac aat gcc ggc aag 816 Ser Asn Ser Arg SerGln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265 270 atc atg gcg ctgacc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 864 Ile Met Ala Leu ThrLys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 275 280 285 ccg ccc gcg gacatt aaa acc aac cgc atc tac cgc atc ctg gag ctg 912 Pro Pro Ala Asp IleLys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 290 295 300 aac ggc tac gaacct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 960 Asn Gly Tyr Glu ProAla Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 305 310 315 320 cag aaa aggttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 1008 Gln Lys Arg PheGly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335 acc acg ggcaag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 1056 Thr Thr Gly LysThr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350 ttc tac ggctgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 1104 Phe Tyr Gly CysVal Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365 tgc gtc gacaag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 1152 Cys Val Asp LysMet Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375 380 aag gtc gtggag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc 1200 Lys Val Val GluSer Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 385 390 395 400 gtg gaccaa aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 1248 Val Asp GlnLys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val 405 410 415 atc gtcacc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 1296 Ile Val ThrSer Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 acc accttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 1344 Thr Thr PheGlu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 gaa ctcacc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 1392 Glu Leu ThrArg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460 gaa gtcaaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 1440 Glu Val LysGlu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 465 470 475 480 gcgcat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 1488 Ala HisGlu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495 cccgat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc 1536 Pro AspAsp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505 510 gcggat cca tcg acg tca gac gcg gaa gga gct ccg gtg gac ttt gcc 1584 Ala AspPro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 515 520 525 gacagg tac caa aac aaa tgt tct cgt cac gcg ggc atg ctt cag atg 1632 Asp ArgTyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu Gln Met 530 535 540 ctgttt ccc tgc aag aca tgc gag aga atg aat cag aat ttc aac att 1680 Leu PhePro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn Phe Asn Ile 545 550 555 560tgc ttc acg cac ggg acg aga gac tgt tca gag tgc ttc ccc ggc gtg 1728 CysPhe Thr His Gly Thr Arg Asp Cys Ser Glu Cys Phe Pro Gly Val 565 570 575tca gaa tct caa ccg gtc gtc aga aag agg acg tat cgg aaa ctc tgt 1776 SerGlu Ser Gln Pro Val Val Arg Lys Arg Thr Tyr Arg Lys Leu Cys 580 585 590gcc att cat cat ctg ctg ggg cgg gct ccc gag att gct tgc tcg gcc 1824 AlaIle His His Leu Leu Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala 595 600 605tgc gat ctg gtc aac gtg gac ctg gat gac tgt gtt tct gag caa taa 1872 CysAsp Leu Val Asn Val Asp Leu Asp Asp Cys Val Ser Glu Gln 610 615 620 5623 PRT AAV-1 5 Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser AspLeu Asp 1 5 10 15 Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Ser TrpVal Ala Glu 20 25 30 Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp LeuAsn Leu Ile 35 40 45 Glu Gln Ala Pro Leu Thr Val Ala Glu Lys Leu Gln ArgAsp Phe Leu 50 55 60 Val Gln Trp Arg Arg Val Ser Lys Ala Pro Glu Ala LeuPhe Phe Val 65 70 75 80 Gln Phe Glu Lys Gly Glu Ser Tyr Phe His Leu HisIle Leu Val Glu 85 90 95 Thr Thr Gly Val Lys Ser Met Val Leu Gly Arg PheLeu Ser Gln Ile 100 105 110 Arg Asp Lys Leu Val Gln Thr Ile Tyr Arg GlyIle Glu Pro Thr Leu 115 120 125 Pro Asn Trp Phe Ala Val Thr Lys Thr ArgAsn Gly Ala Gly Gly Gly 130 135 140 Asn Lys Val Val Asp Glu Cys Tyr IlePro Asn Tyr Leu Leu Pro Lys 145 150 155 160 Thr Gln Pro Glu Leu Gln TrpAla Trp Thr Asn Met Glu Glu Tyr Ile 165 170 175 Ser Ala Cys Leu Asn LeuAla Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 Leu Thr His Val SerGln Thr Gln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 Pro Asn Ser AspAla Pro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215 220 Met Glu LeuVal Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 GlnTrp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265270 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 275280 285 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu290 295 300 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu Gly TrpAla 305 310 315 320 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu PheGly Pro Ala 325 330 335 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile AlaHis Ala Val Pro 340 345 350 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu AsnPhe Pro Phe Asn Asp 355 360 365 Cys Val Asp Lys Met Val Ile Trp Trp GluGlu Gly Lys Met Thr Ala 370 375 380 Lys Val Val Glu Ser Ala Lys Ala IleLeu Gly Gly Ser Lys Val Arg 385 390 395 400 Val Asp Gln Lys Cys Lys SerSer Ala Gln Ile Asp Pro Thr Pro Val 405 410 415 Ile Val Thr Ser Asn ThrAsn Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 Thr Thr Phe Glu HisGln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 Glu Leu Thr ArgArg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460 Glu Val LysGlu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 465 470 475 480 AlaHis Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505510 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 515520 525 Asp Arg Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu Gln Met530 535 540 Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn Phe AsnIle 545 550 555 560 Cys Phe Thr His Gly Thr Arg Asp Cys Ser Glu Cys PhePro Gly Val 565 570 575 Ser Glu Ser Gln Pro Val Val Arg Lys Arg Thr TyrArg Lys Leu Cys 580 585 590 Ala Ile His His Leu Leu Gly Arg Ala Pro GluIle Ala Cys Ser Ala 595 600 605 Cys Asp Leu Val Asn Val Asp Leu Asp AspCys Val Ser Glu Gln 610 615 620 6 1641 DNA AAV-1 CDS (1)..(1638) 6 atgccg ggc ttc tac gag atc gtg atc aag gtg ccg agc gac ctg gac 48 Met ProGly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp Leu Asp 1 5 10 15 gagcac ctg ccg ggc att tct gac tcg ttt gtg agc tgg gtg gcc gag 96 Glu HisLeu Pro Gly Ile Ser Asp Ser Phe Val Ser Trp Val Ala Glu 20 25 30 aag gaatgg gag ctg ccc ccg gat tct gac atg gat ctg aat ctg att 144 Lys Glu TrpGlu Leu Pro Pro Asp Ser Asp Met Asp Leu Asn Leu Ile 35 40 45 gag cag gcaccc ctg acc gtg gcc gag aag ctg cag cgc gac ttc ctg 192 Glu Gln Ala ProLeu Thr Val Ala Glu Lys Leu Gln Arg Asp Phe Leu 50 55 60 gtc caa tgg cgccgc gtg agt aag gcc ccg gag gcc ctc ttc ttt gtt 240 Val Gln Trp Arg ArgVal Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 65 70 75 80 cag ttc gag aagggc gag tcc tac ttc cac ctc cat att ctg gtg gag 288 Gln Phe Glu Lys GlyGlu Ser Tyr Phe His Leu His Ile Leu Val Glu 85 90 95 acc acg ggg gtc aaatcc atg gtg ctg ggc cgc ttc ctg agt cag att 336 Thr Thr Gly Val Lys SerMet Val Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110 agg gac aag ctg gtgcag acc atc tac cgc ggg atc gag ccg acc ctg 384 Arg Asp Lys Leu Val GlnThr Ile Tyr Arg Gly Ile Glu Pro Thr Leu 115 120 125 ccc aac tgg ttc gcggtg acc aag acg cgt aat ggc gcc gga ggg ggg 432 Pro Asn Trp Phe Ala ValThr Lys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140 aac aag gtg gtg gacgag tgc tac atc ccc aac tac ctc ctg ccc aag 480 Asn Lys Val Val Asp GluCys Tyr Ile Pro Asn Tyr Leu Leu Pro Lys 145 150 155 160 act cag ccc gagctg cag tgg gcg tgg act aac atg gag gag tat ata 528 Thr Gln Pro Glu LeuGln Trp Ala Trp Thr Asn Met Glu Glu Tyr Ile 165 170 175 agc gcc tgt ttgaac ctg gcc gag cgc aaa cgg ctc gtg gcg cag cac 576 Ser Ala Cys Leu AsnLeu Ala Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 ctg acc cac gtcagc cag acc cag gag cag aac aag gag aat ctg aac 624 Leu Thr His Val SerGln Thr Gln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 ccc aat tct gacgcg cct gtc atc cgg tca aaa acc tcc gcg cgc tac 672 Pro Asn Ser Asp AlaPro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215 220 atg gag ctg gtcggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag 720 Met Glu Leu Val GlyTrp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 cag tgg atccag gag gac cag gcc tcg tac atc tcc ttc aac gcc gct 768 Gln Trp Ile GlnGlu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255 tcc aac tcgcgg tcc cag atc aag gcc gct ctg gac aat gcc ggc aag 816 Ser Asn Ser ArgSer Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265 270 atc atg gcgctg acc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 864 Ile Met Ala LeuThr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 275 280 285 ccg ccc gcggac att aaa acc aac cgc atc tac cgc atc ctg gag ctg 912 Pro Pro Ala AspIle Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 290 295 300 aac ggc tacgaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 960 Asn Gly Tyr GluPro Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 305 310 315 320 cag aaaagg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 1008 Gln Lys ArgPhe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335 acc acgggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 1056 Thr Thr GlyLys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350 ttc tacggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 1104 Phe Tyr GlyCys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365 tgc gtcgac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 1152 Cys Val AspLys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375 380 aag gtcgtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc 1200 Lys Val ValGlu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 385 390 395 400 gtggac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 1248 Val AspGln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val 405 410 415 atcgtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 1296 Ile ValThr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 accacc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 1344 Thr ThrPhe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 gaactc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 1392 Glu LeuThr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460 gaagtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 1440 Glu ValLys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 465 470 475 480gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 1488 AlaHis Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc 1536 ProAsp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505 510gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gac ttt gcc 1584 AlaAsp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 515 520 525gac agg tat ggc tgc cga tgg tta tct tcc aga ttg gct cga gga caa 1632 AspArg Tyr Gly Cys Arg Trp Leu Ser Ser Arg Leu Ala Arg Gly Gln 530 535 540cct ctc tga 1641 Pro Leu 545 7 546 PRT AAV-1 7 Met Pro Gly Phe Tyr GluIle Val Ile Lys Val Pro Ser Asp Leu Asp 1 5 10 15 Glu His Leu Pro GlyIle Ser Asp Ser Phe Val Ser Trp Val Ala Glu 20 25 30 Lys Glu Trp Glu LeuPro Pro Asp Ser Asp Met Asp Leu Asn Leu Ile 35 40 45 Glu Gln Ala Pro LeuThr Val Ala Glu Lys Leu Gln Arg Asp Phe Leu 50 55 60 Val Gln Trp Arg ArgVal Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 65 70 75 80 Gln Phe Glu LysGly Glu Ser Tyr Phe His Leu His Ile Leu Val Glu 85 90 95 Thr Thr Gly ValLys Ser Met Val Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110 Arg Asp LysLeu Val Gln Thr Ile Tyr Arg Gly Ile Glu Pro Thr Leu 115 120 125 Pro AsnTrp Phe Ala Val Thr Lys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140 AsnLys Val Val Asp Glu Cys Tyr Ile Pro Asn Tyr Leu Leu Pro Lys 145 150 155160 Thr Gln Pro Glu Leu Gln Trp Ala Trp Thr Asn Met Glu Glu Tyr Ile 165170 175 Ser Ala Cys Leu Asn Leu Ala Glu Arg Lys Arg Leu Val Ala Gln His180 185 190 Leu Thr His Val Ser Gln Thr Gln Glu Gln Asn Lys Glu Asn LeuAsn 195 200 205 Pro Asn Ser Asp Ala Pro Val Ile Arg Ser Lys Thr Ser AlaArg Tyr 210 215 220 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile ThrSer Glu Lys 225 230 235 240 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr IleSer Phe Asn Ala Ala 245 250 255 Ser Asn Ser Arg Ser Gln Ile Lys Ala AlaLeu Asp Asn Ala Gly Lys 260 265 270 Ile Met Ala Leu Thr Lys Ser Ala ProAsp Tyr Leu Val Gly Pro Ala 275 280 285 Pro Pro Ala Asp Ile Lys Thr AsnArg Ile Tyr Arg Ile Leu Glu Leu 290 295 300 Asn Gly Tyr Glu Pro Ala TyrAla Gly Ser Val Phe Leu Gly Trp Ala 305 310 315 320 Gln Lys Arg Phe GlyLys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335 Thr Thr Gly LysThr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350 Phe Tyr GlyCys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365 Cys ValAsp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375 380 LysVal Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 385 390 395400 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val 405410 415 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser420 425 430 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe LysPhe 435 440 445 Glu Leu Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val ThrLys Gln 450 455 460 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His ValThr Glu Val 465 470 475 480 Ala His Glu Phe Tyr Val Arg Lys Gly Gly AlaAsn Lys Arg Pro Ala 485 490 495 Pro Asp Asp Ala Asp Lys Ser Glu Pro LysArg Ala Cys Pro Ser Val 500 505 510 Ala Asp Pro Ser Thr Ser Asp Ala GluGly Ala Pro Val Asp Phe Ala 515 520 525 Asp Arg Tyr Gly Cys Arg Trp LeuSer Ser Arg Leu Ala Arg Gly Gln 530 535 540 Pro Leu 545 8 1200 DNA AAV-1CDS (1)..(1197) 8 atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acctcc gag aag 48 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr SerGlu Lys 1 5 10 15 cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttcaac gcc gct 96 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe AsnAla Ala 20 25 30 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat gccggc aag 144 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala GlyLys 35 40 45 atc atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc cccgct 192 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala50 55 60 ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg240 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 6570 75 80 aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc288 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 8590 95 cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc336 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 100105 110 acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc384 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 115120 125 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat432 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130135 140 tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc480 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145150 155 160 aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtgcgc 528 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg165 170 175 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc cccgtg 576 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val180 185 190 atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aacagc 624 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser195 200 205 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaattt 672 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe210 215 220 gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aagcag 720 Glu Leu Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln225 230 235 240 gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg accgag gtg 768 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr GluVal 245 250 255 gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa agaccc gcc 816 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg ProAla 260 265 270 ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccctca gtc 864 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro SerVal 275 280 285 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gacttt gcc 912 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp PheAla 290 295 300 gac agg tac caa aac aaa tgt tct cgt cac gcg ggc atg cttcag atg 960 Asp Arg Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu GlnMet 305 310 315 320 ctg ttt ccc tgc aag aca tgc gag aga atg aat cag aatttc aac att 1008 Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn PheAsn Ile 325 330 335 tgc ttc acg cac ggg acg aga gac tgt tca gag tgc ttcccc ggc gtg 1056 Cys Phe Thr His Gly Thr Arg Asp Cys Ser Glu Cys Phe ProGly Val 340 345 350 tca gaa tct caa ccg gtc gtc aga aag agg acg tat cggaaa ctc tgt 1104 Ser Glu Ser Gln Pro Val Val Arg Lys Arg Thr Tyr Arg LysLeu Cys 355 360 365 gcc att cat cat ctg ctg ggg cgg gct ccc gag att gcttgc tcg gcc 1152 Ala Ile His His Leu Leu Gly Arg Ala Pro Glu Ile Ala CysSer Ala 370 375 380 tgc gat ctg gtc aac gtg gac ctg gat gac tgt gtt tctgag caa taa 1200 Cys Asp Leu Val Asn Val Asp Leu Asp Asp Cys Val Ser GluGln 385 390 395 9 399 PRT AAV-1 9 Met Glu Leu Val Gly Trp Leu Val AspArg Gly Ile Thr Ser Glu Lys 1 5 10 15 Gln Trp Ile Gln Glu Asp Gln AlaSer Tyr Ile Ser Phe Asn Ala Ala 20 25 30 Ser Asn Ser Arg Ser Gln Ile LysAla Ala Leu Asp Asn Ala Gly Lys 35 40 45 Ile Met Ala Leu Thr Lys Ser AlaPro Asp Tyr Leu Val Gly Pro Ala 50 55 60 Pro Pro Ala Asp Ile Lys Thr AsnArg Ile Tyr Arg Ile Leu Glu Leu 65 70 75 80 Asn Gly Tyr Glu Pro Ala TyrAla Gly Ser Val Phe Leu Gly Trp Ala 85 90 95 Gln Lys Arg Phe Gly Lys ArgAsn Thr Ile Trp Leu Phe Gly Pro Ala 100 105 110 Thr Thr Gly Lys Thr AsnIle Ala Glu Ala Ile Ala His Ala Val Pro 115 120 125 Phe Tyr Gly Cys ValAsn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130 135 140 Cys Val Asp LysMet Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145 150 155 160 Lys ValVal Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 165 170 175 ValAsp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val 180 185 190Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 195 200205 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 210215 220 Glu Leu Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln225 230 235 240 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val ThrGlu Val 245 250 255 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn LysArg Pro Ala 260 265 270 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg AlaCys Pro Ser Val 275 280 285 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly AlaPro Val Asp Phe Ala 290 295 300 Asp Arg Tyr Gln Asn Lys Cys Ser Arg HisAla Gly Met Leu Gln Met 305 310 315 320 Leu Phe Pro Cys Lys Thr Cys GluArg Met Asn Gln Asn Phe Asn Ile 325 330 335 Cys Phe Thr His Gly Thr ArgAsp Cys Ser Glu Cys Phe Pro Gly Val 340 345 350 Ser Glu Ser Gln Pro ValVal Arg Lys Arg Thr Tyr Arg Lys Leu Cys 355 360 365 Ala Ile His His LeuLeu Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala 370 375 380 Cys Asp Leu ValAsn Val Asp Leu Asp Asp Cys Val Ser Glu Gln 385 390 395 10 969 DNA AAV-1CDS (1)..(966) 10 atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acctcc gag aag 48 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr SerGlu Lys 1 5 10 15 cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttcaac gcc gct 96 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe AsnAla Ala 20 25 30 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat gccggc aag 144 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala GlyLys 35 40 45 atc atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc cccgct 192 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala50 55 60 ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg240 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 6570 75 80 aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc288 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 8590 95 cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc336 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 100105 110 acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc384 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 115120 125 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat432 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130135 140 tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc480 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145150 155 160 aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtgcgc 528 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg165 170 175 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc cccgtg 576 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val180 185 190 atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aacagc 624 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser195 200 205 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaattt 672 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe210 215 220 gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aagcag 720 Glu Leu Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln225 230 235 240 gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg accgag gtg 768 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr GluVal 245 250 255 gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa agaccc gcc 816 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg ProAla 260 265 270 ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccctca gtc 864 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro SerVal 275 280 285 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gacttt gcc 912 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp PheAla 290 295 300 gac agg tat ggc tgc cga tgg tta tct tcc aga ttg gct cgagga caa 960 Asp Arg Tyr Gly Cys Arg Trp Leu Ser Ser Arg Leu Ala Arg GlyGln 305 310 315 320 cct ctc tga 969 Pro Leu 11 322 PRT AAV-1misc_feature (943)..(944) minor splice site 11 Met Glu Leu Val Gly TrpLeu Val Asp Arg Gly Ile Thr Ser Glu Lys 1 5 10 15 Gln Trp Ile Gln GluAsp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 20 25 30 Ser Asn Ser Arg SerGln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 35 40 45 Ile Met Ala Leu ThrLys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 50 55 60 Pro Pro Ala Asp IleLys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 65 70 75 80 Asn Gly Tyr GluPro Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 85 90 95 Gln Lys Arg PheGly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 100 105 110 Thr Thr GlyLys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 115 120 125 Phe TyrGly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130 135 140 CysVal Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145 150 155160 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 165170 175 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val180 185 190 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly AsnSer 195 200 205 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met PheLys Phe 210 215 220 Glu Leu Thr Arg Arg Leu Glu His Asp Phe Gly Lys ValThr Lys Gln 225 230 235 240 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln AspHis Val Thr Glu Val 245 250 255 Ala His Glu Phe Tyr Val Arg Lys Gly GlyAla Asn Lys Arg Pro Ala 260 265 270 Pro Asp Asp Ala Asp Lys Ser Glu ProLys Arg Ala Cys Pro Ser Val 275 280 285 Ala Asp Pro Ser Thr Ser Asp AlaGlu Gly Ala Pro Val Asp Phe Ala 290 295 300 Asp Arg Tyr Gly Cys Arg TrpLeu Ser Ser Arg Leu Ala Arg Gly Gln 305 310 315 320 Pro Leu 12 2211 DNAAAV-1 CDS (1)..(2208) 12 atg gct gcc gat ggt tat ctt cca gat tgg ctc gaggac aac ctc tct 48 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu AspAsn Leu Ser 1 5 10 15 gag ggc att cgc gag tgg tgg gac ttg aaa cct ggagcc ccg aag ccc 96 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly AlaPro Lys Pro 20 25 30 aaa gcc aac cag caa aag cag gac gac ggc cgg ggt ctggtg ctt cct 144 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu ValLeu Pro 35 40 45 ggc tac aag tac ctc gga ccc ttc aac gga ctc gac aag ggggag ccc 192 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly GluPro 50 55 60 gtc aac gcg gcg gac gca gcg gcc ctc gag cac gac aag gcc tacgac 240 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80 cag cag ctc aaa gcg ggt gac aat ccg tac ctg cgg tat aac cacgcc 288 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala85 90 95 gac gcc gag ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggc336 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100105 110 aac ctc ggg cga gca gtc ttc cag gcc aag aag cgg gtt ctc gaa cct384 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115120 125 ctc ggt ctg gtt gag gaa ggc gct aag acg gct cct gga aag aaa cgt432 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130135 140 ccg gta gag cag tcg cca caa gag cca gac tcc tcc tcg ggc atc ggc480 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145150 155 160 aag aca ggc cag cag ccc gct aaa aag aga ctc aat ttt ggt cagact 528 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr165 170 175 ggc gac tca gag tca gtc ccc gat cca caa cct ctc gga gaa cctcca 576 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro180 185 190 gca acc ccc gct gct gtg gga cct act aca atg gct tca ggc ggtggc 624 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly195 200 205 gca cca atg gca gac aat aac gaa ggc gcc gac gga gtg ggt aatgcc 672 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala210 215 220 tca gga aat tgg cat tgc gat tcc aca tgg ctg ggc gac aga gtcatc 720 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile225 230 235 240 acc acc agc acc cgc acc tgg gcc ttg ccc acc tac aat aaccac ctc 768 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn HisLeu 245 250 255 tac aag caa atc tcc agt gct tca acg ggg gcc agc aac gacaac cac 816 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp AsnHis 260 265 270 tac ttc ggc tac agc acc ccc tgg ggg tat ttt gat ttc aacaga ttc 864 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn ArgPhe 275 280 285 cac tgc cac ttt tca cca cgt gac tgg cag cga ctc atc aacaac aat 912 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn AsnAsn 290 295 300 tgg gga ttc cgg ccc aag aga ctc aac ttc aaa ctc ttc aacatc caa 960 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn IleGln 305 310 315 320 gtc aag gag gtc acg acg aat gat ggc gtc aca acc atcgct aat aac 1008 Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile AlaAsn Asn 325 330 335 ctt acc agc acg gtt caa gtc ttc tcg gac tcg gag taccag ctt ccg 1056 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr GlnLeu Pro 340 345 350 tac gtc ctc ggc tct gcg cac cag ggc tgc ctc cct ccgttc ccg gcg 1104 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro PhePro Ala 355 360 365 gac gtg ttc atg att ccg caa tac ggc tac ctg acg ctcaac aat ggc 1152 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu AsnAsn Gly 370 375 380 agc caa gcc gtg gga cgt tca tcc ttt tac tgc ctg gaatat ttc cct 1200 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu TyrPhe Pro 385 390 395 400 tct cag atg ctg aga acg ggc aac aac ttt acc ttcagc tac acc ttt 1248 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe SerTyr Thr Phe 405 410 415 gag gaa gtg cct ttc cac agc agc tac gcg cac agccag agc ctg gac 1296 Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser GlnSer Leu Asp 420 425 430 cgg ctg atg aat cct ctc atc gac caa tac ctg tattac ctg aac aga 1344 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr TyrLeu Asn Arg 435 440 445 act caa aat cag tcc gga agt gcc caa aac aag gacttg ctg ttt agc 1392 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp LeuLeu Phe Ser 450 455 460 cgt ggg tct cca gct ggc atg tct gtt cag ccc aaaaac tgg cta cct 1440 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys AsnTrp Leu Pro 465 470 475 480 gga ccc tgt tat cgg cag cag cgc gtt tct aaaaca aaa aca gac aac 1488 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys ThrLys Thr Asp Asn 485 490 495 aac aac agc aat ttt acc tgg act ggt gct tcaaaa tat aac ctc aat 1536 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser LysTyr Asn Leu Asn 500 505 510 ggg cgt gaa tcc atc atc aac cct ggc act gctatg gcc tca cac aaa 1584 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala MetAla Ser His Lys 515 520 525 gac gac gaa gac aag ttc ttt ccc atg agc ggtgtc atg att ttt gga 1632 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly ValMet Ile Phe Gly 530 535 540 aaa gag agc gcc gga gct tca aac act gca ttggac aat gtc atg att 1680 Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu AspAsn Val Met Ile 545 550 555 560 aca gac gaa gag gaa att aaa gcc act aaccct gtg gcc acc gaa aga 1728 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn ProVal Ala Thr Glu Arg 565 570 575 ttt ggg acc gtg gca gtc aat ttc cag agcagc agc aca gac cct gcg 1776 Phe Gly Thr Val Ala Val Asn Phe Gln Ser SerSer Thr Asp Pro Ala 580 585 590 acc gga gat gtg cat gct atg gga gca ttacct ggc atg gtg tgg caa 1824 Thr Gly Asp Val His Ala Met Gly Ala Leu ProGly Met Val Trp Gln 595 600 605 gat aga gac gtg tac ctg cag ggt ccc atttgg gcc aaa att cct cac 1872 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile TrpAla Lys Ile Pro His 610 615 620 aca gat gga cac ttt cac ccg tct cct cttatg ggc ggc ttt gga ctc 1920 Thr Asp Gly His Phe His Pro Ser Pro Leu MetGly Gly Phe Gly Leu 625 630 635 640 aag aac ccg cct cct cag atc ctc atcaaa aac acg cct gtt cct gcg 1968 Lys Asn Pro Pro Pro Gln Ile Leu Ile LysAsn Thr Pro Val Pro Ala 645 650 655 aat cct ccg gcg gag ttt tca gct acaaag ttt gct tca ttc atc acc 2016 Asn Pro Pro Ala Glu Phe Ser Ala Thr LysPhe Ala Ser Phe Ile Thr 660 665 670 caa tac tcc aca gga caa gtg agt gtggaa att gaa tgg gag ctg cag 2064 Gln Tyr Ser Thr Gly Gln Val Ser Val GluIle Glu Trp Glu Leu Gln 675 680 685 aaa gaa aac agc aag cgc tgg aat cccgaa gtg cag tac aca tcc aat 2112 Lys Glu Asn Ser Lys Arg Trp Asn Pro GluVal Gln Tyr Thr Ser Asn 690 695 700 tat gca aaa tct gcc aac gtt gat tttact gtg gac aac aat gga ctt 2160 Tyr Ala Lys Ser Ala Asn Val Asp Phe ThrVal Asp Asn Asn Gly Leu 705 710 715 720 tat act gag cct cgc ccc att ggcacc cgt tac ctt acc cgt ccc ctg 2208 Tyr Thr Glu Pro Arg Pro Ile Gly ThrArg Tyr Leu Thr Arg Pro Leu 725 730 735 taa 2211 13 736 PRT AAV-1 13 MetAla Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 7580 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 9095 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys LysArg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser GlyIle Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu AsnPhe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln ProLeu Gly Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr ThrMet Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu GlyAla Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp SerThr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg ThrTrp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile SerSer Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270 Tyr Phe Gly TyrSer Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys HisPhe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp GlyPhe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330335 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn AsnGly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu TyrPhe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr PheSer Tyr Thr Phe 405 410 415 Glu Glu Val Pro Phe His Ser Ser Tyr Ala HisSer Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln TyrLeu Tyr Tyr Leu Asn Arg 435 440 445 Thr Gln Asn Gln Ser Gly Ser Ala GlnAsn Lys Asp Leu Leu Phe Ser 450 455 460 Arg Gly Ser Pro Ala Gly Met SerVal Gln Pro Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg GlnGln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495 Asn Asn Ser Asn PheThr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu SerIle Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525 Asp Asp GluAsp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540 Lys GluSer Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile 545 550 555 560Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570575 Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580585 590 Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile ProHis 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly PheGly Leu 625 630 635 640 Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn ThrPro Val Pro Ala 645 650 655 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys PheAla Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val GluIle Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn ProGlu Val Gln Tyr Thr Ser Asn 690 695 700 Tyr Ala Lys Ser Ala Asn Val AspPhe Thr Val Asp Asn Asn Gly Leu 705 710 715 720 Tyr Thr Glu Pro Arg ProIle Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735 14 1800 DNA AAV-1CDS (1)..(1797) 14 acg gct cct gga aag aaa cgt ccg gta gag cag tcg ccacaa gag cca 48 Thr Ala Pro Gly Lys Lys Arg Pro Val Glu Gln Ser Pro GlnGlu Pro 1 5 10 15 gac tcc tcc tcg ggc atc ggc aag aca ggc cag cag cccgct aaa aag 96 Asp Ser Ser Ser Gly Ile Gly Lys Thr Gly Gln Gln Pro AlaLys Lys 20 25 30 aga ctc aat ttt ggt cag act ggc gac tca gag tca gtc cccgat cca 144 Arg Leu Asn Phe Gly Gln Thr Gly Asp Ser Glu Ser Val Pro AspPro 35 40 45 caa cct ctc gga gaa cct cca gca acc ccc gct gct gtg gga cctact 192 Gln Pro Leu Gly Glu Pro Pro Ala Thr Pro Ala Ala Val Gly Pro Thr50 55 60 aca atg gct tca ggc ggt ggc gca cca atg gca gac aat aac gaa ggc240 Thr Met Ala Ser Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly 6570 75 80 gcc gac gga gtg ggt aat gcc tca gga aat tgg cat tgc gat tcc aca288 Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp His Cys Asp Ser Thr 8590 95 tgg ctg ggc gac aga gtc atc acc acc agc acc cgc acc tgg gcc ttg336 Trp Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu 100105 110 ccc acc tac aat aac cac ctc tac aag caa atc tcc agt gct tca acg384 Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr 115120 125 ggg gcc agc aac gac aac cac tac ttc ggc tac agc acc ccc tgg ggg432 Gly Ala Ser Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 130135 140 tat ttt gat ttc aac aga ttc cac tgc cac ttt tca cca cgt gac tgg480 Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 145150 155 160 cag cga ctc atc aac aac aat tgg gga ttc cgg ccc aag aga ctcaac 528 Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn165 170 175 ttc aaa ctc ttc aac atc caa gtc aag gag gtc acg acg aat gatggc 576 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Asp Gly180 185 190 gtc aca acc atc gct aat aac ctt acc agc acg gtt caa gtc ttctcg 624 Val Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Ser195 200 205 gac tcg gag tac cag ctt ccg tac gtc ctc ggc tct gcg cac cagggc 672 Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly210 215 220 tgc ctc cct ccg ttc ccg gcg gac gtg ttc atg att ccg caa tacggc 720 Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly225 230 235 240 tac ctg acg ctc aac aat ggc agc caa gcc gtg gga cgt tcatcc ttt 768 Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser SerPhe 245 250 255 tac tgc ctg gaa tat ttc cct tct cag atg ctg aga acg ggcaac aac 816 Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly AsnAsn 260 265 270 ttt acc ttc agc tac acc ttt gag gaa gtg cct ttc cac agcagc tac 864 Phe Thr Phe Ser Tyr Thr Phe Glu Glu Val Pro Phe His Ser SerTyr 275 280 285 gcg cac agc cag agc ctg gac cgg ctg atg aat cct ctc atcgac caa 912 Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile AspGln 290 295 300 tac ctg tat tac ctg aac aga act caa aat cag tcc gga agtgcc caa 960 Tyr Leu Tyr Tyr Leu Asn Arg Thr Gln Asn Gln Ser Gly Ser AlaGln 305 310 315 320 aac aag gac ttg ctg ttt agc cgt ggg tct cca gct ggcatg tct gtt 1008 Asn Lys Asp Leu Leu Phe Ser Arg Gly Ser Pro Ala Gly MetSer Val 325 330 335 cag ccc aaa aac tgg cta cct gga ccc tgt tat cgg cagcag cgc gtt 1056 Gln Pro Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln GlnArg Val 340 345 350 tct aaa aca aaa aca gac aac aac aac agc aat ttt acctgg act ggt 1104 Ser Lys Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe Thr TrpThr Gly 355 360 365 gct tca aaa tat aac ctc aat ggg cgt gaa tcc atc atcaac cct ggc 1152 Ala Ser Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile AsnPro Gly 370 375 380 act gct atg gcc tca cac aaa gac gac gaa gac aag ttcttt ccc atg 1200 Thr Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe PhePro Met 385 390 395 400 agc ggt gtc atg att ttt gga aaa gag agc gcc ggagct tca aac act 1248 Ser Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly AlaSer Asn Thr 405 410 415 gca ttg gac aat gtc atg att aca gac gaa gag gaaatt aaa gcc act 1296 Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu IleLys Ala Thr 420 425 430 aac cct gtg gcc acc gaa aga ttt ggg acc gtg gcagtc aat ttc cag 1344 Asn Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala ValAsn Phe Gln 435 440 445 agc agc agc aca gac cct gcg acc gga gat gtg catgct atg gga gca 1392 Ser Ser Ser Thr Asp Pro Ala Thr Gly Asp Val His AlaMet Gly Ala 450 455 460 tta cct ggc atg gtg tgg caa gat aga gac gtg tacctg cag ggt ccc 1440 Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr LeuGln Gly Pro 465 470 475 480 att tgg gcc aaa att cct cac aca gat gga cacttt cac ccg tct cct 1488 Ile Trp Ala Lys Ile Pro His Thr Asp Gly His PheHis Pro Ser Pro 485 490 495 ctt atg ggc ggc ttt gga ctc aag aac ccg cctcct cag atc ctc atc 1536 Leu Met Gly Gly Phe Gly Leu Lys Asn Pro Pro ProGln Ile Leu Ile 500 505 510 aaa aac acg cct gtt cct gcg aat cct ccg gcggag ttt tca gct aca 1584 Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Ala GluPhe Ser Ala Thr 515 520 525 aag ttt gct tca ttc atc acc caa tac tcc acagga caa gtg agt gtg 1632 Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr GlyGln Val Ser Val 530 535 540 gaa att gaa tgg gag ctg cag aaa gaa aac agcaag cgc tgg aat ccc 1680 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser LysArg Trp Asn Pro 545 550 555 560 gaa gtg cag tac aca tcc aat tat gca aaatct gcc aac gtt gat ttt 1728 Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys SerAla Asn Val Asp Phe 565 570 575 act gtg gac aac aat gga ctt tat act gagcct cgc ccc att ggc acc 1776 Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu ProArg Pro Ile Gly Thr 580 585 590 cgt tac ctt acc cgt ccc ctg taa 1800 ArgTyr Leu Thr Arg Pro Leu 595 15 599 PRT AAV-1 15 Thr Ala Pro Gly Lys LysArg Pro Val Glu Gln Ser Pro Gln Glu Pro 1 5 10 15 Asp Ser Ser Ser GlyIle Gly Lys Thr Gly Gln Gln Pro Ala Lys Lys 20 25 30 Arg Leu Asn Phe GlyGln Thr Gly Asp Ser Glu Ser Val Pro Asp Pro 35 40 45 Gln Pro Leu Gly GluPro Pro Ala Thr Pro Ala Ala Val Gly Pro Thr 50 55 60 Thr Met Ala Ser GlyGly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly 65 70 75 80 Ala Asp Gly ValGly Asn Ala Ser Gly Asn Trp His Cys Asp Ser Thr 85 90 95 Trp Leu Gly AspArg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu 100 105 110 Pro Thr TyrAsn Asn His Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr 115 120 125 Gly AlaSer Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 130 135 140 TyrPhe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 145 150 155160 Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn 165170 175 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Asp Gly180 185 190 Val Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val PheSer 195 200 205 Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala HisGln Gly 210 215 220 Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile ProGln Tyr Gly 225 230 235 240 Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala ValGly Arg Ser Ser Phe 245 250 255 Tyr Cys Leu Glu Tyr Phe Pro Ser Gln MetLeu Arg Thr Gly Asn Asn 260 265 270 Phe Thr Phe Ser Tyr Thr Phe Glu GluVal Pro Phe His Ser Ser Tyr 275 280 285 Ala His Ser Gln Ser Leu Asp ArgLeu Met Asn Pro Leu Ile Asp Gln 290 295 300 Tyr Leu Tyr Tyr Leu Asn ArgThr Gln Asn Gln Ser Gly Ser Ala Gln 305 310 315 320 Asn Lys Asp Leu LeuPhe Ser Arg Gly Ser Pro Ala Gly Met Ser Val 325 330 335 Gln Pro Lys AsnTrp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val 340 345 350 Ser Lys ThrLys Thr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly 355 360 365 Ala SerLys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly 370 375 380 ThrAla Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro Met 385 390 395400 Ser Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr 405410 415 Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr420 425 430 Asn Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala Val Asn PheGln 435 440 445 Ser Ser Ser Thr Asp Pro Ala Thr Gly Asp Val His Ala MetGly Ala 450 455 460 Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr LeuGln Gly Pro 465 470 475 480 Ile Trp Ala Lys Ile Pro His Thr Asp Gly HisPhe His Pro Ser Pro 485 490 495 Leu Met Gly Gly Phe Gly Leu Lys Asn ProPro Pro Gln Ile Leu Ile 500 505 510 Lys Asn Thr Pro Val Pro Ala Asn ProPro Ala Glu Phe Ser Ala Thr 515 520 525 Lys Phe Ala Ser Phe Ile Thr GlnTyr Ser Thr Gly Gln Val Ser Val 530 535 540 Glu Ile Glu Trp Glu Leu GlnLys Glu Asn Ser Lys Arg Trp Asn Pro 545 550 555 560 Glu Val Gln Tyr ThrSer Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe 565 570 575 Thr Val Asp AsnAsn Gly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr 580 585 590 Arg Tyr LeuThr Arg Pro Leu 595 16 1605 DNA AAV-1 CDS (1)..(1602) 16 atg gct tca ggcggt ggc gca cca atg gca gac aat aac gaa ggc gcc 48 Met Ala Ser Gly GlyGly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 gac gga gtg ggtaat gcc tca gga aat tgg cat tgc gat tcc aca tgg 96 Asp Gly Val Gly AsnAla Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 25 30 ctg ggc gac aga gtcatc acc acc agc acc cgc acc tgg gcc ttg ccc 144 Leu Gly Asp Arg Val IleThr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45 acc tac aat aac cac ctctac aag caa atc tcc agt gct tca acg ggg 192 Thr Tyr Asn Asn His Leu TyrLys Gln Ile Ser Ser Ala Ser Thr Gly 50 55 60 gcc agc aac gac aac cac tacttc ggc tac agc acc ccc tgg ggg tat 240 Ala Ser Asn Asp Asn His Tyr PheGly Tyr Ser Thr Pro Trp Gly Tyr 65 70 75 80 ttt gat ttc aac aga ttc cactgc cac ttt tca cca cgt gac tgg cag 288 Phe Asp Phe Asn Arg Phe His CysHis Phe Ser Pro Arg Asp Trp Gln 85 90 95 cga ctc atc aac aac aat tgg ggattc cgg ccc aag aga ctc aac ttc 336 Arg Leu Ile Asn Asn Asn Trp Gly PheArg Pro Lys Arg Leu Asn Phe 100 105 110 aaa ctc ttc aac atc caa gtc aaggag gtc acg acg aat gat ggc gtc 384 Lys Leu Phe Asn Ile Gln Val Lys GluVal Thr Thr Asn Asp Gly Val 115 120 125 aca acc atc gct aat aac ctt accagc acg gtt caa gtc ttc tcg gac 432 Thr Thr Ile Ala Asn Asn Leu Thr SerThr Val Gln Val Phe Ser Asp 130 135 140 tcg gag tac cag ctt ccg tac gtcctc ggc tct gcg cac cag ggc tgc 480 Ser Glu Tyr Gln Leu Pro Tyr Val LeuGly Ser Ala His Gln Gly Cys 145 150 155 160 ctc cct ccg ttc ccg gcg gacgtg ttc atg att ccg caa tac ggc tac 528 Leu Pro Pro Phe Pro Ala Asp ValPhe Met Ile Pro Gln Tyr Gly Tyr 165 170 175 ctg acg ctc aac aat ggc agccaa gcc gtg gga cgt tca tcc ttt tac 576 Leu Thr Leu Asn Asn Gly Ser GlnAla Val Gly Arg Ser Ser Phe Tyr 180 185 190 tgc ctg gaa tat ttc cct tctcag atg ctg aga acg ggc aac aac ttt 624 Cys Leu Glu Tyr Phe Pro Ser GlnMet Leu Arg Thr Gly Asn Asn Phe 195 200 205 acc ttc agc tac acc ttt gaggaa gtg cct ttc cac agc agc tac gcg 672 Thr Phe Ser Tyr Thr Phe Glu GluVal Pro Phe His Ser Ser Tyr Ala 210 215 220 cac agc cag agc ctg gac cggctg atg aat cct ctc atc gac caa tac 720 His Ser Gln Ser Leu Asp Arg LeuMet Asn Pro Leu Ile Asp Gln Tyr 225 230 235 240 ctg tat tac ctg aac agaact caa aat cag tcc gga agt gcc caa aac 768 Leu Tyr Tyr Leu Asn Arg ThrGln Asn Gln Ser Gly Ser Ala Gln Asn 245 250 255 aag gac ttg ctg ttt agccgt ggg tct cca gct ggc atg tct gtt cag 816 Lys Asp Leu Leu Phe Ser ArgGly Ser Pro Ala Gly Met Ser Val Gln 260 265 270 ccc aaa aac tgg cta cctgga ccc tgt tat cgg cag cag cgc gtt tct 864 Pro Lys Asn Trp Leu Pro GlyPro Cys Tyr Arg Gln Gln Arg Val Ser 275 280 285 aaa aca aaa aca gac aacaac aac agc aat ttt acc tgg act ggt gct 912 Lys Thr Lys Thr Asp Asn AsnAsn Ser Asn Phe Thr Trp Thr Gly Ala 290 295 300 tca aaa tat aac ctc aatggg cgt gaa tcc atc atc aac cct ggc act 960 Ser Lys Tyr Asn Leu Asn GlyArg Glu Ser Ile Ile Asn Pro Gly Thr 305 310 315 320 gct atg gcc tca cacaaa gac gac gaa gac aag ttc ttt ccc atg agc 1008 Ala Met Ala Ser His LysAsp Asp Glu Asp Lys Phe Phe Pro Met Ser 325 330 335 ggt gtc atg att tttgga aaa gag agc gcc gga gct tca aac act gca 1056 Gly Val Met Ile Phe GlyLys Glu Ser Ala Gly Ala Ser Asn Thr Ala 340 345 350 ttg gac aat gtc atgatt aca gac gaa gag gaa att aaa gcc act aac 1104 Leu Asp Asn Val Met IleThr Asp Glu Glu Glu Ile Lys Ala Thr Asn 355 360 365 cct gtg gcc acc gaaaga ttt ggg acc gtg gca gtc aat ttc cag agc 1152 Pro Val Ala Thr Glu ArgPhe Gly Thr Val Ala Val Asn Phe Gln Ser 370 375 380 agc agc aca gac cctgcg acc gga gat gtg cat gct atg gga gca tta 1200 Ser Ser Thr Asp Pro AlaThr Gly Asp Val His Ala Met Gly Ala Leu 385 390 395 400 cct ggc atg gtgtgg caa gat aga gac gtg tac ctg cag ggt ccc att 1248 Pro Gly Met Val TrpGln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile 405 410 415 tgg gcc aaa attcct cac aca gat gga cac ttt cac ccg tct cct ctt 1296 Trp Ala Lys Ile ProHis Thr Asp Gly His Phe His Pro Ser Pro Leu 420 425 430 atg ggc ggc tttgga ctc aag aac ccg cct cct cag atc ctc atc aaa 1344 Met Gly Gly Phe GlyLeu Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys 435 440 445 aac acg cct gttcct gcg aat cct ccg gcg gag ttt tca gct aca aag 1392 Asn Thr Pro Val ProAla Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys 450 455 460 ttt gct tca ttcatc acc caa tac tcc aca gga caa gtg agt gtg gaa 1440 Phe Ala Ser Phe IleThr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu 465 470 475 480 att gaa tgggag ctg cag aaa gaa aac agc aag cgc tgg aat ccc gaa 1488 Ile Glu Trp GluLeu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu 485 490 495 gtg cag tacaca tcc aat tat gca aaa tct gcc aac gtt gat ttt act 1536 Val Gln Tyr ThrSer Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr 500 505 510 gtg gac aacaat gga ctt tat act gag cct cgc ccc att ggc acc cgt 1584 Val Asp Asn AsnGly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg 515 520 525 tac ctt acccgt ccc ctg taa 1605 Tyr Leu Thr Arg Pro Leu 530 17 534 PRT AAV-1 17 MetAla Ser Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala 1 5 10 15Asp Gly Val Gly Asn Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 25 30Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly 50 55 60Ala Ser Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr 65 70 7580 Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln 85 9095 Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe 100105 110 Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val115 120 125 Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe SerAsp 130 135 140 Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His GlnGly Cys 145 150 155 160 Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile ProGln Tyr Gly Tyr 165 170 175 Leu Thr Leu Asn Asn Gly Ser Gln Ala Val GlyArg Ser Ser Phe Tyr 180 185 190 Cys Leu Glu Tyr Phe Pro Ser Gln Met LeuArg Thr Gly Asn Asn Phe 195 200 205 Thr Phe Ser Tyr Thr Phe Glu Glu ValPro Phe His Ser Ser Tyr Ala 210 215 220 His Ser Gln Ser Leu Asp Arg LeuMet Asn Pro Leu Ile Asp Gln Tyr 225 230 235 240 Leu Tyr Tyr Leu Asn ArgThr Gln Asn Gln Ser Gly Ser Ala Gln Asn 245 250 255 Lys Asp Leu Leu PheSer Arg Gly Ser Pro Ala Gly Met Ser Val Gln 260 265 270 Pro Lys Asn TrpLeu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser 275 280 285 Lys Thr LysThr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala 290 295 300 Ser LysTyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr 305 310 315 320Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro Met Ser 325 330335 Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala 340345 350 Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn355 360 365 Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala Val Asn Phe GlnSer 370 375 380 Ser Ser Thr Asp Pro Ala Thr Gly Asp Val His Ala Met GlyAla Leu 385 390 395 400 Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr LeuGln Gly Pro Ile 405 410 415 Trp Ala Lys Ile Pro His Thr Asp Gly His PheHis Pro Ser Pro Leu 420 425 430 Met Gly Gly Phe Gly Leu Lys Asn Pro ProPro Gln Ile Leu Ile Lys 435 440 445 Asn Thr Pro Val Pro Ala Asn Pro ProAla Glu Phe Ser Ala Thr Lys 450 455 460 Phe Ala Ser Phe Ile Thr Gln TyrSer Thr Gly Gln Val Ser Val Glu 465 470 475 480 Ile Glu Trp Glu Leu GlnLys Glu Asn Ser Lys Arg Trp Asn Pro Glu 485 490 495 Val Gln Tyr Thr SerAsn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr 500 505 510 Val Asp Asn AsnGly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg 515 520 525 Tyr Leu ThrArg Pro Leu 530 18 4681 DNA AAV-2 18 ttggccactc cctctctgcg cgctcgctcgctcactgagg ccgggcgacc aaaggtcgcc 60 cgacgcccgg gctttgcccg ggcggcctcagtgagcgagc gagcgcgcag agagggagtg 120 gccaactcca tcactagggg ttcctggaggggtggagtcg tgacgtgaat tacgtcatag 180 ggttagggag gtcctgtatt agaggtcacgtgagtgtttt gcgacatttt gcgacaccat 240 gtggtcacgc tgggtattta agcccgagtgagcacgcagg gtctccattt tgaagcggga 300 ggtttgaacg cgcagccgcc atgccggggttttacgagat tgtgattaag gtccccagcg 360 accttgacgg gcatctgccc ggcatttctgacagctttgt gaactgggtg gccgagaagg 420 aatgggagtt gccgccagat tctgacatggatctgaatct gattgagcag gcacccctga 480 ccgtggccga gaagctgcag cgcgactttctgacggaatg gcgccgtgtg agtaaggccc 540 cggaggccct tttctttgtg caatttgagaagggagagag ctacttccac atgcacgtgc 600 tcgtggaaac caccggggtg aaatccatggttttgggacg tttcctgagt cagattcgcg 660 aaaaactgat tcagagaatt taccgcgggatcgagccgac tttgccaaac tggttcgcgg 720 tcacaaagac cagaaatggc gccggaggcgggaacaaggt ggtggatgag tgctacatcc 780 ccaattactt gctccccaaa acccagcctgagctccagtg ggcgtggact aatatggaac 840 agtatttaag cgcctgtttg aatctcacggagcgtaaacg gttggtggcg cagcatctga 900 cgcacgtgtc gcagacgcag gagcagaacaaagagaatca gaatcccaat tctgatgcgc 960 cggtgatcag atcaaaaact tcagccaggtacatggagct ggtcgggtgg ctcgtggaca 1020 aggggattac ctcggagaag cagtggatccaggaggacca ggcctcatac atctccttca 1080 atgcggcctc caactcgcgg tcccaaatcaaggctgcctt ggacaatgcg ggaaagatta 1140 tgagcctgac taaaaccgcc cccgactacctggtgggcca gcagcccgtg gaggacattt 1200 ccagcaatcg gatttataaa attttggaactaaacgggta cgatccccaa tatgcggctt 1260 ccgtctttct gggatgggcc acgaaaaagttcggcaagag gaacaccatc tggctgtttg 1320 ggcctgcaac taccgggaag accaacatcgcggaggccat agcccacact gtgcccttct 1380 acgggtgcgt aaactggacc aatgagaactttcccttcaa cgactgtgtc gacaagatgg 1440 tgatctggtg ggaggagggg aagatgaccgccaaggtcgt ggagtcggcc aaagccattc 1500 tcggaggaag caaggtgcgc gtggaccagaaatgcaagtc ctcggcccag atagacccga 1560 ctcccgtgat cgtcacctcc aacaccaacatgtgcgccgt gattgacggg aactcaacga 1620 ccttcgaaca ccagcagccg ttgcaagaccggatgttcaa atttgaactc acccgccgtc 1680 tggatcatga ctttgggaag gtcaccaagcaggaagtcaa agactttttc cggtgggcaa 1740 aggatcacgt ggttgaggtg gagcatgaattctacgtcaa aaagggtgga gccaagaaaa 1800 gacccgcccc cagtgacgca gatataagtgagcccaaacg ggtgcgcgag tcagttgcgc 1860 agccatcgac gtcagacgcg gaagcttcgatcaactacgc agacaggtac caaaacaaat 1920 gttctcgtca cgtgggcatg aatctgatgctgtttccctg cagacaatgc gagagaatga 1980 atcagaattc aaatatctgc ttcactcacggacagaaaga ctgtttagag tgctttcccg 2040 tgtcagaatc tcaaccggtt tctgtcgtcaaaaaggcgta tcagaaactg tgctacattc 2100 atcatatcat gggaaaggtg ccagacgcttgcactgcctg cgatctggtc aatgtggatt 2160 tggatgactg catctttgaa caataaatgatttaaatcag gtatggctgc cgatggttat 2220 cttccagatt ggctcgagga cactctctctgaaggaataa gacagtggtg gaagctcaaa 2280 cctggcccac caccaccaaa gcccgcagagcggcataagg acgacagcag gggtcttgtg 2340 cttcctgggt acaagtacct cggacccttcaacggactcg acaagggaga gccggtcaac 2400 gaggcagacg ccgcggccct cgagcacgacaaagcctacg accggcagct cgacagcgga 2460 gacaacccgt acctcaagta caaccacgccgacgcggagt ttcaggagcg ccttaaagaa 2520 gatacgtctt ttgggggcaa cctcggacgagcagtcttcc aggcgaaaaa gagggttctt 2580 gaacctctcg gcctggttga ggaacctgttaagacggctc cgggaaaaaa gaggccggta 2640 gagcactctc ctgtggagcc agactcctcctcgggaaccg gaaagccggg ccagcagcct 2700 gcaagaaaaa gattgaattt tggtcagactggagacgcag actcagtacc tgacccccag 2760 cctctcggac agccaccagc agccccctctggtctgggaa ctaatacgat ggctacaggc 2820 agtggcgcac caatggcaga caataacgagggcgccgacg gagtgggtaa ttcctccgga 2880 aattggcatt gcgattccac atggatgggcgacagagtca tcaccaccag cacccgaacc 2940 tgggccctgc ccacctacaa caaccacctctacaaacaaa tttccagcca atcaggagcc 3000 tcgaacgaca atcactactt tggctacagcaccccttggg ggtattttga cttcaacaga 3060 ttccactgcc acttttcacc acgtgactggcaaagactca tcaacaacaa ctggggattc 3120 cgacccaaga gactcaactt caacctctttaacattcaag tcaaagaggt cacgcagaat 3180 gacggtacga cgacgattgc caataaccttaccagcacgg ttcaggtgtt tactgactcg 3240 gagtaccagc tcccgtacgt cctcggctcggcgcatcaag gatgcctccc gccgttccca 3300 gcagacgtct tcatggtgcc acagtatggatacctcaccc tgaacaacgg gagtcaggca 3360 gtaggacgct cttcatttta ctgcctggagtactttcctt ctcagatgct gcgtaccgga 3420 aacaacttta ccttcagcta cacttttgaggacgttcctt tccacagcag ctacgctcac 3480 agccagagtc tggaccgtct catgaatcctctcatcgacc agtacctgta ttacttgagc 3540 agaacaaaca ctccaagtgg aaccaccacgcagtcaaggc ttcagttttc tcaggcccca 3600 gccagtgaca ttcgggacca gtctaggaactggcttcctg gaccctgtta ccgccagcag 3660 cgagtatgaa agacatctgc ggataacaacaacagtgaat actcgtggac tggagctacc 3720 aagtaccacc tcaatggcag agactctctggtgaatccgg ggcccgccat ggcaagccac 3780 aaggacgatg aagaaaagtt ttttcctcagagcggggttc tcatctttgg gaagcaaggc 3840 tcagagaaaa caaatgtgaa cattgaaaaggtcatgatta cagacgaaga ggaaatccca 3900 acaaccaatc ccgtggctac ggagcagtatggttctgtat ctaccaacct ccagagaggc 3960 aacagacaag cagctaccgc agatgtcaacacacaaggcg ttcttccagg catggtctgg 4020 caggacagag atgtgtacct tcaggggcccatctgggcaa agattccaca cacggacgga 4080 cattttcacc cctctcccct catgggtggattcggactta aacaccctcc tccacagatt 4140 ctcatcaaga acaccccggt acctgcgaatccttcgacca ccttcagtgc ggcaaagttt 4200 gcttccttca tcacacagta ctccacgggacacggtcagc gtggagatcg agtgggagct 4260 gcagaacgaa aacagcaaac gctggaatcccgaaattcag tacacttcca actacaacaa 4320 gtctgttaat cgtggacttt accgtggatactaatggcgt gtattcagag cctcgcccca 4380 ttggcaccag atacctgact cgtaatctgtaattgcttgt taatcaataa accgtttaat 4440 tcgtttcagt tgaactttgg tctctgcgtatttctttctt atctagtttc catggctacg 4500 tagataagta gcatggcggg ttaatcattaactacaagga acccctagtg atggagttgg 4560 ccactccctc tctgcgcgct cgctcgctcactgaggccgg gcgaccaaag gtcgcccgac 4620 gcccgggctt tgccccggcg gcctcagtgagcgagcgagc gcgcagagag ggagtgggca 4680 a 4681 19 4683 DNA AAV-6 19ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag 180ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300ggtttgaacg cgcagcgcca tgccggggtt ttacgagatt gtgattaagg tccccagcga 360ccttgacgag catctgcccg gcatttctga cagctttgtg aactgggtgg ccgagaagga 420atgggagttg ccgccagatt ctgacatgga tctgaatctg attgagcagg cacccctgac 480cgtggccgag aagctgcagc gcgacttcct ggtccactgg cgccgcgtga gtaaggcccc 540ggaggccctc ttctttgttc agttcgagaa gggcgagtcc tacttccacc tccatattct 600ggtggagacc acgggggtca aatccatggt gctgggccgc ttcctgagtc agattagcga 660caagctggtg cagaccatct accgcgggat cgagccgacc ctgcccaact ggttcgcggt 720gaccaagacg cgtaatggcg ccggaggggg gaacaaggtg gtggacgagt gctacatccc 780caactacctc ctgcccaaga ctcagcccga gctgcagtgg gcgtggacta acatggagga 840gtatataagc gcgtgtttaa acctggccga gcgcaaacgg ctcgtggcgc acgacctgac 900ccacgtcagc cagacccagg agcagaacaa ggagaatctg aaccccaatt ctgacgcgcc 960tgtcatccgg tcaaaaacct ccgcacgcta catggagctg gtcgggtggc tggtggaccg 1020gggcatcacc tccgagaagc agtggatcca ggaggaccag gcctcgtaca tctccttcaa 1080cgccgcctcc aactcgcggt cccagatcaa ggccgctctg gacaatgccg gcaagatcat 1140ggcgctgacc aaatccgcgc ccgactacct ggtaggcccc gctccgcccg ccgacattaa 1200aaccaaccgc atttaccgca tcctggagct gaacggctac gaccctgcct acgccggctc 1260cgtctttctc ggctgggccc agaaaaggtt cggaaaacgc aacaccatct ggctgtttgg 1320gccggccacc acgggcaaga ccaacatcgc ggaagccatc gcccacgccg tgcccttcta 1380cggctgcgtc aactggacca atgagaactt tcccttcaac gattgcgtcg acaagatggt 1440gatctggtgg gaggagggca agatgacggc caaggtcgtg gagtccgcca aggccattct 1500cggcggcagc aaggtgcgcg tggaccaaaa gtgcaagtcg tccgcccaga tcgatcccac 1560ccccgtgatc gtcacctcca acaccaacat gtgcgccgtg attgacggga acagcaccac 1620cttcgagcac cagcagccgt tgcaggaccg gatgttcaaa tttgaactca cccgccgtct 1680ggagcatgac tttggcaagg tgacaaagca ggaagtcaaa gagttcttcc gctgggcgca 1740ggatcacgtg accgaggtgg cgcatgagtt ctacgtcaga aagggtggag ccaacaacag 1800acccgccccc gatgacgcgg ataaaagcga gcccaagcgg gcctgcccct cagtcgcgga 1860tccatcgacg tcagacgcgg aaggagctcc ggtggacttt gccgacaggt accaaaacaa 1920atgttctcgt cacgcgggca tgcttcagat gctgtttccc tgcaaaacat gcgagagaat 1980gaatcagaat ttcaacattt gcttcacgca cgggaccaga gactgttcag aatgtttccc 2040cggcgtgtca gaatctcaac cggtcgtcag aaagaggacg tatcggaaac tctgtgccat 2100tcatcatctg ctggggcggg ctcccgagat tgcttgctcg gcctgcgatc tggtcaacgt 2160ggatctggat gactgtgttt ctgagcaata aatgacttaa accaggtatg gctgccgatg 2220gttatcttcc agattggctc gaggacaacc tctctgaggg cattcggcag tggtgggact 2280tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacgac ggccggggtc 2340tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg 2400tcaacgcggc ggatgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag 2460cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc 2520aagaagatac gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagaggg 2580ttctcgaacc ttttggtctg gttgaggaag gtgctaagac ggctcctgga aagaaacgtc 2640cggtagagca gtcgccacaa gagccagact cctcctcggg cattggcaag acaggccagc 2700agcccgctaa aaagagactc aattttggtc agactggcga ctcagagtca gtccccgacc 2760cacaacctct cggagaacct ccagcaaccc ccgctgctgt gggacctact acaatggctt 2820caggcggtgg cgcaccaatg gcagacaata acgaaggcgc cgacggagtg ggtaatgcct 2880caggaaattg gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc 2940gaacatgggc cttgcccacc tataacaacc acctctacaa gcaaatctcc agtgcttcaa 3000cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg tattttgatt 3060tcaacagatt ccactgccat ttctcaccac gtgactggca gcgactcatc aacaacaatt 3120ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc aaggaggtca 3180cgacgaatga tggcgtcacg accatcgcta ataaccttac cagcacggtt caagtcttgt 3240cggactcgga gtaccagttc ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc 3300cgttcccggc ggacgtgttc atgattccgc agtacggcta cctaacgctc aacaatggca 3360gccaggcagt gggacgctca tccttttact gcctggaata tttcccatcg cagatgctga 3420gaacgggcaa taactttacc ttcagctaca ccttcgagga cgtgcctttc cacagcagct 3480acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag tacctgtatt 3540acctgaacag aactcacaat cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc 3600gtgggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttacc 3660ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt acctggactg 3720gtgcttcaaa atataacctt aatgggcgtg aatctataat caaccctggc actgctatgg 3780cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg atttttggaa 3840aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca gacgaagagg 3900aaatcaaagc cactaacccc gtggccaccg aaagatttgg gactgtggca gtcaatctcc 3960agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc ttacctggaa 4020tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa attcctcaca 4080cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag cacccgcctc 4140ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag ttttcggcta 4200caaagtttgc ttcattcatc acccagtatt ccacaggaca agtgagcgtg gagattgaat 4260gggagctgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat acatctaact 4320atgcaaaatc tgccaacgtt gatttcactg tggacaacaa tggactttat actgagcctc 4380gccccattgg cacccgttac ctcacccgtc ccctgtaatt gtgtgttaat caataaaccg 4440gttaattcgt gtcagttgaa ctttggtctc atgtccttat tatcttatct ggtcaccata 4500gcaaccggtt acacattaac tgcttagttg cgcttcgcga atacccctag tgatggagtt 4560gcccactccc tctatgcgcg ctcgctcgct cggtggggcc ggcagagcag agctctgccg 4620tctgcggacc tttggtccgc aggccccacc gagcgagcga gcgcgcatag agggagtggc 4680caa 4683 20 16 DNA rep binding motif 20 gctcgctcgc tcgctg 16

What is claimed is:
 1. A method of delivering a heterologous nucleicacid to at least one muscle cell in a mammalian subject, comprising: (a)providing at least one recombinant adeno-associated virus (rAAV) virion,said rAAV virion comprising an AAV-6 capsid and a heterologous nucleicacid operably linked to expression control elements; and (b)administering said rAAV virions to said muscle cell, whereby expressionof said heterologous nucleic acid provides for a therapeutic effect. 2.The method of claim 1, wherein said heterologous nucleic acid is a geneencoding a protein.
 3. The method of claim 1, wherein said heterologousnucleic acid is an antisense RNA.
 4. The method of claim 2, wherein saidprotein is a secreted protein.
 5. The method of claim 4, wherein saidsecreted protein is selected from the group consisting of cytokines,growth factors, and differentiation factors.
 6. The method of claim 2,wherein said protein is alpha1-antitrypsin or erthryopoietin.
 7. Themethod of claim 1, wherein said administering of said rAAV virions is byway of direct injection to said muscle cell of said mammalian subject.8. The method of claim 7, wherein said muscle cell is a skeletal musclecell.
 9. The method of claim 1, wherein said administering of said rAAVvirions is by way of administration to a vascular conduit of saidmammalian subject.
 10. The method of claim 9, wherein said vascularconduit is a vein.